scholarly journals Data-Driven Prediction of CRISPR-Based Transcription Regulation for Programmable Control of Metabolic Flux

2017 ◽  
Author(s):  
Jiayuan Sheng ◽  
Weihua Guo ◽  
Christine Ash ◽  
Brendan Freitas ◽  
Mitchell Paoletti ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Metabolic Flux ◽  
Target Genes ◽  
Product Yield ◽  
Fine Tuning ◽  
Data Driven ◽  
Gene Expressions ◽  
Guide Rnas ◽  
Directional Control ◽  
Transcription Regulators

AbstractMultiplex and multi-directional control of metabolic pathways is crucial for metabolic engineering to improve product yield of fuels, chemicals, and pharmaceuticals. To achieve this goal, artificial transcriptional regulators such as CRISPR-based transcription regulators have been developed to specifically activate or repress genes of interest. Here, we found that by deploying guide RNAs to target on DNA sites at different locations of genetic cassettes, we could use just one synthetic CRISPR-based transcriptional regulator to simultaneously activate and repress gene expressions. By using the pairwise datasets of guide RNAs and gene expressions, we developed a data-driven predictive model to rationally design this system for fine-tuning expression of target genes. We demonstrated that this system could achieve programmable control of metabolic fluxes when using yeast to produce versatile chemicals. We anticipate that this master CRISPR-based transcription regulator will be a valuable addition to the synthetic biology toolkit for metabolic engineering, speeding up the “design-build-test” cycle in industrial biomanufacturing as well as generating new biological insights on the fates of eukaryotic cells.

scholarly journals Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

2019 ◽  
Vol 48 (2) ◽  
pp. 996-1009 ◽  
Author(s):  
Yaokang Wu ◽  
Taichi Chen ◽  
Yanfeng Liu ◽  
Rongzhen Tian ◽  
Xueqin Lv ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Target Genes ◽  
Fine Tuning ◽  
Dual Control ◽  
Genetic Circuits ◽  
Dynamic Regulation ◽  
Batch Bioreactor ◽  
Acetoin Production

Abstract Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

scholarly journals Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ying Li ◽  
He Xian ◽  
Ya Xu ◽  
Yuan Zhu ◽  
Zhijie Sun ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Conversion Rate ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Fine Tuning ◽  
High Yield ◽  
Glycolytic Flux ◽  
Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

scholarly journals New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

2020 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
Jaiana Malabarba ◽  
Elisabeth Chevreau ◽  
Nicolas Dousset ◽  
Florian Veillet ◽  
Julie Moizan ◽  
...  
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Acetolactate Synthase ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Adventitious Regeneration ◽  
Base Editing ◽  
Perennial Plants ◽  
Guide Rnas ◽  
Albino Phenotype

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

scholarly journals Manipulation of the ATP pool as a tool for metabolic engineering

2015 ◽  
Vol 43 (6) ◽  
pp. 1140-1145 ◽  
Author(s):  
Oliver Hädicke ◽  
Steffen Klamt
Keyword(s):  
Metabolic Engineering ◽  
Product Yield ◽  
Volumetric Productivity ◽  
Special Focus ◽  
Process Performance ◽  
Performance Parameters ◽  
Cofactor Engineering ◽  
Atp Pool ◽  
Fine Tune

Cofactor engineering has been long identified as a valuable tool for metabolic engineering. Besides interventions targeting the pools of redox cofactors, many studies addressed the adenosine pools of microorganisms. In this mini-review, we discuss interventions that manipulate the availability of ATP with a special focus on ATP wasting strategies. We discuss the importance to fine-tune the ATP yield along a production pathway to balance process performance parameters like product yield and volumetric productivity.

scholarly journals Exploiting the GTEx resources to decipher the mechanisms at GWAS loci

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Alvaro N. Barbeira ◽  
◽  
Rodrigo Bonazzola ◽  
Eric R. Gamazon ◽  
Yanyu Liang ◽  
...  
Keyword(s):  
Complex Traits ◽  
Target Genes ◽  
Genome Wide Association Study ◽  
Data Driven ◽  
Functional Interpretation ◽  
Transcriptome Regulation ◽  
Genome Wide ◽  
Causal Genes ◽  
Dose Dependent ◽  
Single Approach

AbstractThe resources generated by the GTEx consortium offer unprecedented opportunities to advance our understanding of the biology of human diseases. Here, we present an in-depth examination of the phenotypic consequences of transcriptome regulation and a blueprint for the functional interpretation of genome-wide association study-discovered loci. Across a broad set of complex traits and diseases, we demonstrate widespread dose-dependent effects of RNA expression and splicing. We develop a data-driven framework to benchmark methods that prioritize causal genes and find no single approach outperforms the combination of multiple approaches. Using colocalization and association approaches that take into account the observed allelic heterogeneity of gene expression, we propose potential target genes for 47% (2519 out of 5385) of the GWAS loci examined.

scholarly journals Constraint-based metabolic control analysis for rational strain engineering

2020 ◽  
Author(s):  
Sophia Tsouka ◽  
Meric Ataman ◽  
Tuure Hameri ◽  
Ljubisa Miskovic ◽  
Vassily Hatzimanikatis
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Metabolic Control ◽  
Metabolic Flux ◽  
Strain Engineering ◽  
Metabolic Control Analysis ◽  
Physiological Data ◽  
Response Analysis ◽  
Control Analysis ◽  
Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

scholarly journals A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fillip Port ◽  
Claudia Strein ◽  
Mona Stricker ◽  
Benedikt Rauscher ◽  
Florian Heigwer ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
High Efficiency ◽  
Direct Sequencing ◽  
Fine Tuning ◽  
Genetic Screens ◽  
Efficient Gene ◽  
Crispr Screen ◽  
High Gene ◽  
Multicellular Organisms

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

Abstract WMP80: Micro RNA-Therapy in Murine Thromboembolic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Kumar Vaibhav ◽  
Shannon Williams ◽  
Sumbul Fatima ◽  
Babak Baban ◽  
Krishnan M Dhandapani ◽  
...  
Keyword(s):  
Target Genes ◽  
Infarct Volume ◽  
Protein Translation ◽  
Micro Rna ◽  
Thromboembolic Stroke ◽  
Gene Expressions ◽  
Mirna Array ◽  
Micro Rnas ◽  
Pai 1

Background: Micro RNAs (miRNAs) could target multiple mRNAs, repressing the protein translation. We report acute changes in humoral miRNAome in a murine thromboembolic stroke model (eMCAo), and demonstrate the benefits of miRNA therapy in improving cerebral blood flow (CBF). Methods: Non-biased micro RNA (miRNA) array and bioinformatics analysis was performed in plasma collected at 4h post-eMCAo from male mice (C57/B6, 16-weeks). Individual PCR for miRNAs was also performed in brain tissues at 24h post-eMCAo. Moreover, frozen human plasma samples collected at ~4.5h post-stroke were also used for miRNA analysis. Finally, the miRNA mimic that was predicted to target genes of our interest was also tested in vivo and in vitro . Results: Principal component analysis (PCA) of the miRNA-array showed ~68% variance in the humoral miRNAome 4h after eMCAo in mice, and a significant change in Stroke vs. Sham groups (Cut off value >2 fold; p<0.05). Of interest, the hairpin precursor of miR-449b was downregulated (~2.35 fold, p<0.05) at 4h post-eMCAo, while the mature miR-449b was also significantly reduced at 24h post-eMCAo. Mature miR-449b was significantly reduced in human stroke plasma, too. In human brain endothelial cells, miR-449b mimic downregulated gene expressions of both plasminogen activator inhibitor (PAI-1) and alpha 2- antiplasmin (α-AP) only in hypoxia but not during normoxia. Therefore, we finally tested the cholesterol-conjugated miR-449b mimic in the murine eMCAo model. Pre-treatment with miR-449b mimic (8 mg/kg bwt) increased the absolute CBF and reduced edema (as determined by MRI), and also improved the neurological outcomes and reduced % infarct volume (p<0.05). Results: The miR-449b mimic could be a possible therapy to suppress aberrant gene expressions of PAI-1 and α-AP, which will allow more spontaneous reperfusion and benefits from low dose tPA.

scholarly journals Interplay between miRNAs and host genes and their role in cancer

2019 ◽  
Vol 18 (4) ◽  
pp. 255-266 ◽  
Author(s):  
Baohong Liu ◽  
Yu Shyr ◽  
Jianping Cai ◽  
Qi Liu
Keyword(s):  
Tumor Suppressors ◽  
Target Genes ◽  
Epithelial Mesenchymal Transition ◽  
Fine Tuning ◽  
Data Sets ◽  
Protein Coding ◽  
Mesenchymal Transition ◽  
Damage Repair ◽  
Cancer Types ◽  
Host Genes

Abstract MicroRNAs (miRNAs) are small endogenous non-coding functional RNAs that post-transcriptionally regulate gene expression. They play essential roles in nearly all biological processes including cell development and differentiation, DNA damage repair, cell death as well as intercellular communication. They are highly involved in cancer, acting as tumor suppressors and/or promoters to modulate cell proliferation, epithelial-mesenchymal transition and tumor invasion and metastasis. Recent studies have shown that more than half of miRNAs are located within protein-coding or non-coding genes. Intragenic miRNAs and their host genes either share the promoter or have independent transcription. Meanwhile, miRNAs work as partners or antagonists of their host genes by fine-tuning their target genes functionally associated with host genes. This review outlined the complicated relationship between intragenic miRNAs and host genes. Focusing on miRNAs known as oncogenes or tumor suppressors in specific cancer types, it studied co-expression relationships between these miRNAs and host genes in the cancer types using TCGA data sets, which validated previous findings and revealed common, tumor-specific and even subtype-specific patterns. These observations will help understand the function of intragenic miRNAs and further develop miRNA therapeutics in cancer.

scholarly journals Small-molecule inhibitor targeting orphan nuclear receptor COUP-TFII for prostate cancer treatment

2020 ◽  
Vol 6 (18) ◽  
pp. eaaz8031 ◽  
Author(s):  
Leiming Wang ◽  
Chiang-Min Cheng ◽  
Jun Qin ◽  
Mafei Xu ◽  
Chung-Yang Kao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Nuclear Receptor ◽  
High Throughput Screening ◽  
Target Gene ◽  
Target Genes ◽  
Orphan Nuclear Receptor ◽  
Prostate Cancer Treatment ◽  
Molecule Inhibitor ◽  
Transcription Regulators ◽  
Xenograft Models

The orphan nuclear receptor COUP-TFII is expressed at a low level in adult tissues, but its expression is increased and shown to promote progression of multiple diseases, including prostate cancer, heart failure, and muscular dystrophy. Suppression of COUP-TFII slows disease progression, making it an intriguing therapeutic target. Here, we identified a potent and specific COUP-TFII inhibitor through high-throughput screening. The inhibitor specifically suppressed COUP-TFII activity to regulate its target genes. Mechanistically, the inhibitor directly bound to the COUP-TFII ligand-binding domain and disrupted COUP-TFII interaction with transcription regulators, including FOXA1, thus repressing COUP-TFII activity on target gene regulation. Through blocking COUP-TFII’s oncogenic activity in prostate cancer, the inhibitor efficiently exerted a potent antitumor effect in xenograft mouse models and patient-derived xenograft models. Our study identified a potent and specific COUP-TFII inhibitor that may be useful for the treatment of prostate cancer and possibly other diseases.

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