scholarly journals Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)

2020 ◽  
Vol 2 ◽  
Author(s):  
Charlotte De Bruyn ◽  
Tom Ruttink ◽  
Tom Eeckhaut ◽  
Thomas Jacobs ◽  
Ellen De Keyser ◽  
...  
Keyword(s):  
Genome Editing ◽  
Sesquiterpene Lactones ◽  
Amplicon Sequencing ◽  
Industrial Applications ◽  
Cichorium Intybus ◽  
Biosynthesis Pathway ◽  
Regenerated Plants ◽  
Nucleotide Insertion ◽  
Protoplast Transfection ◽  
Vector Targeting

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.

scholarly journals Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications

2021 ◽  
Vol 9 (8) ◽  
pp. 1642
Author(s):  
Dorothee Tegtmeier ◽  
Sabine Hurka ◽  
Sanja Mihajlovic ◽  
Maren Bodenschatz ◽  
Stephanie Schlimbach ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Amplicon Sequencing ◽  
Industrial Applications ◽  
Culture Collection ◽  
Rrna Gene ◽  
Organic Substrates ◽  
Culturable Bacteria ◽  
Black Soldier Fly ◽  
Culture Independent ◽  
Culture Dependent

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

scholarly journals Kasani beej (Cichorium intybus): Ayurvedic View, Folk View, Phytochemistry and Modern Therapeutic Uses

2021 ◽  
Vol 8 (2) ◽  
pp. 114-125
Author(s):  
Shailja Choudhary ◽  
Hemlata Kaurav ◽  
Gitika Chaudhary
Keyword(s):  
Medicinal Plant ◽  
Arid Regions ◽  
Sesquiterpene Lactones ◽  
Cichorium Intybus ◽  
Vegetable Crop ◽  
Therapeutic Uses ◽  
Traditional Remedy ◽  
Coffee Substitute ◽  
Phytochemical Constituents ◽  
Asteraceae Family

Cichorium intybus is commonly known as Chicory, kasani. It is a recognized medicinal plant that belongs to the Asteraceae family. This plant is a well-known traditional herb used in various medicinal systems like Ayurveda, Unani and Siddha medicinal system to treat variety of diseases ranging from wounds to diabetes. The species is native to the European region (Mediterranean region) but can also grow in the temperate and semi-arid regions. The juice extracted from this plant is used as a traditional remedy to cure uterus cancer and tumors. The plant is well-known for its roots that are used as an additive in coffee as it provides bitterness in taste without the caffeine. It is also reported that the roots of the plant contain 40% inulin and acts as an anti-diabetic agent. Historically, the plant was cultivated by ancient Egyptians as a medicinal plant, vegetable crop, coffee substitute and also used as animal forage. Cichorium intybus contains various phytochemical constituents mainly sesquiterpene lactones, caffeic acid derivatives, inulin flavonoids, alkaloids, phenols, steroids terpenoids and many more. The reported pharmacological properties of the C. intybus plant include hepatoprotective, anti-diabetic, anti-cancerous, anti-inflammatory, analgesic, cardiovascular, antioxidant, antimicrobial, anthelmintic, antimalarial, anti-allergic and gastroprotective activities. In this review article, the medicinal and ayurvedic importance of C. intybus plant along with its phytochemicals are briefly explained.

scholarly journals A silencer repressing redundant enhancer activities revealed by deleting endogenouscis-regulatory element ofebonyinDrosophila melanogaster

2021 ◽  
Author(s):  
Noriyoshi Akiyama ◽  
Shoma Sato ◽  
Kentaro M. Tanaka ◽  
Takaomi Sakai ◽  
Aya Takahashi
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Editing ◽  
Expression Pattern ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Biosynthesis Pathway ◽  
Melanin Biosynthesis ◽  
Pigment Synthesis ◽  
Endogenous Expression ◽  
Spatiotemporal Regulation

AbstractThe spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns inDrosophilaare formed by the deposition of different pigments synthesized in the developing epidermis and the role ofcis-regulatory elements (CREs) of melanin biosynthesis pathway-related genes is well-characterized. These CREs typically exhibit modular arrangement in the regulatory region of the gene with each enhancer regulating a specific spatiotemporal expression of the gene. However, recent studies have suggested that multiple enhancers of a number of developmental genes as well as those ofyellow(involved in dark pigment synthesis) exhibit redundant activities. Here we report the redundant enhancer activities in thecis-regulatory region of another gene in the melanin biosynthesis pathway,ebony, in the developing epidermis ofDrosophila melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the endogenous primary epidermis enhancer (priEE) by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of amCherry-taggedebonyallele was examined in the thoracic and abdominal segments. The expression level ofebonyin the priEE-deleted strains was similar to that of the control strain, indicating the presence of redundant enhancer activities that drive the broad expression ofebonyin the developing epidermis. Additionally, the priEE fragment contained a silencer that suppressesebonyexpression in the dorsal midline of the abdominal tergites, which is necessary for the development of the subgenusSophophora-specific dark pigmentation patterns along the midline. The endogenous expression pattern ofebonyin the priEE-deleted strains and the reporter assay examining the autonomous activity of the priEE fragment indicated that the silencer is involved in repressing the activities of both proximal and distant enhancers. These results suggest that multiple silencers are dispensable in the regulatory system of a relatively stable taxonomic character. The prevalence of other redundant enhancers and silencers in the genome can be investigated using a similar approach.Author summaryGenes are expressed at the right timing and place to give rise to diverse phenotypes. The spatiotemporal regulation is usually achieved through the coordinated activities of transcription-activating and transcription-repressing proteins that bind to the DNA sequences called enhancers and silencers, respectively, located near the target gene. Most studies identified the locations of enhancers by examining the ability of the sequence fragments to regulate the expression of fused reporters. Various short enhancers have been identified using this approach. This study employed an alternative approach in which the previously identified enhancer that regulates expression ofebony(a gene involved in body color formation) was deleted in a fruitfly,Drosophila melanogaster, using the genome-editing technique. The knockout of this enhancer did not affect the transcription level of the gene to a large extent. This indicated the presence of transcription-activating elements with redundant functions outside the deleted enhancer. Additionally, the transcription ofebonyat the midline of the abdomen, which is repressed in the normal flies, were derepressed in the enhancer-deleted flies, which indicated that the deleted enhancer fragment contained a silencer that negatively regulates multiple enhancer activities in a spatially restricted manner.

scholarly journals Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vijaydev Ganesan ◽  
Michael Spagnuolo ◽  
Ayushi Agrawal ◽  
Spencer Smith ◽  
Difeng Gao ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Gene Editing ◽  
Molecular Genetic ◽  
Industrial Applications ◽  
Cell Factory ◽  
Fuel Feed ◽  
Renewable Chemicals ◽  
Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

Sesquiterpene Lactones from Cichorium intybus

2011 ◽  
Vol 66 ◽  
pp. 0729 ◽  
Author(s):  
S. Shah ◽  
Z. Ali ◽  
A. Malik ◽  
I. A. Khan ◽  
S. Saied
Keyword(s):  
Sesquiterpene Lactones ◽  
Cichorium Intybus

scholarly journals Relationship between Levels of Sesquiterpene Lactones in Chicory and Sensory Evaluation

1998 ◽  
Vol 123 (2) ◽  
pp. 326-329 ◽  
Author(s):  
Angeline M. Peters ◽  
Aart van Amerongen
Keyword(s):  
Regression Analysis ◽  
Pilot Study ◽  
Linear Regression ◽  
Sensory Evaluation ◽  
Sesquiterpene Lactones ◽  
Linear Regression Analysis ◽  
Cichorium Intybus ◽  
Flavor Intensity ◽  
The Relationship

In this pilot study, we investigated the relationship between levels of bitter sesquiterpene lactones and sensory evaluation of chicory (Cichorium intybus L.). The levels of two bitter sesquiterpene lactones—lactucopicrin and lactucin-like sesquiterpene lactones—were measured by ELISA in raw and cooked chicory samples from several cultivars. Data were compared with the results of a sensory evaluation on the flavor attributes bitterness, typical chicory flavor, and total flavor intensity of identical chicory samples. Linear regression analysis demonstrated that the levels of lactucin-like sesquiterpene lactones were significantly related to bitterness (P = 0.006) and total flavor intensity (P = 0.03) attributes in raw chicory samples. When cooked chicory samples were evaluated, the levels of lactucin-like sesquiterpene lactones were significantly related to bitterness (P = 0.002), typical chicory flavor (P < 0.001), and total flavor intensity (P = 0.009) attributes, while lactucopicrin levels were related to bitterness (P = 0.002) only. These results show that the ELISA can be useful to predict flavor attributes in chicory.

scholarly journals The Global State of Genome Editing

2018 ◽  
Author(s):  
Geoffrey H. Siwo
Keyword(s):  
Genome Editing ◽  
Industrial Applications ◽  
Plant Diseases ◽  
Great Promise ◽  
Literature Mining ◽  
Full Potential ◽  
Scientific Publications ◽  
Gender Biases ◽  
Duchene Muscular Dystrophy ◽  
The Impact

AbstractGenome editing technologies hold great promise in fundamental biomedical research, development of treatments for animal and plant diseases, and engineering biological organisms for food and industrial applications. Therefore, a global understanding of the growth of the field is needed to identify challenges, opportunities and biases that could shape the impact of the technology. To address this, this work applies automated literature mining of scientific publications on genome editing in the past year to infer research trends in 2 key genome editing technologies-CRISPR/Cas systems and TALENs. The study finds that genome editing research is disproportionately distributed between and within countries, with researchers in the US and China accounting for 50% of authors in the field whereas countries across Africa are underrepresented. Furthermore, genome editing research is also disproportionately being explored on diseases such as cancer, Duchene Muscular Dystrophy, sickle cell disease and malaria. Gender biases are also evident in genome editing research with considerably fewer women as principal investigators. The results of this study suggest that automated mining of scientific literature could help identify biases in genome editing research as a means to mitigate future inequalities and tap the full potential of the technology.

scholarly journals Molecular Abiotic Stress Tolerans Strategies: From Genetic Engineering to Genome Editing Era

2020 ◽  
Author(s):  
Sinan Meriç ◽  
Alp Ayan ◽  
Çimen Atak
Keyword(s):  
Abiotic Stress ◽  
Genetic Engineering ◽  
Stress Tolerance ◽  
Genome Editing ◽  
Target Genes ◽  
Abiotic Stress Tolerance ◽  
Industrial Applications ◽  
Genomic Engineering ◽  
Transporter Protein ◽  
Abiotic Stress Factors

In last decades, plants were increasingly subjected to multiple environmental abiotic stress factors as never before due to their stationary nature. Excess urbanization following the intense industrial applications introduced combinations of abiotic stresses as heat, drought, salinity, heavy metals etc. to plants in various intensities. Technological advancements brought novel biotechnological tools to the abiotic stress tolerance area as an alternative to time and money consuming traditional crop breeding activities as well as they brought vast majority of the problem themselves. Discoveries of single gene (as osmoprotectant, detoxyfying enzyme, transporter protein genes etc.) and multi gene (biomolecule synthesis, heat shock protein, regulatory transcription factor and signal transduction genes etc.) targets through functional genomic approaches identified abiotic stress responsive genes through EST based cDNA micro and macro arrays. In nowadays, genetic engineering and genome editing tools are present to transfer genes among different species and modify these target genes in site specific, even single nuclotide specific manner. This present chapter will evaluate genomic engineering approaches and applications targeting these abiotic stress tolerance responsive mechanisms as well as future prospects of genome editing applications in this field.

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