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https://www.readbyqxmd.com/read/29199103/diversion-of-the-long-chain-acyl-acp-pool-in-synechocystis-to-fatty-alcohols-through-crispri-repression-of-the-essential-phosphate-acyltransferase-plsx
#1
Danuta Kaczmarzyk, Ivana Cengic, Lun Yao, Elton P Hudson
Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways...
November 30, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/29181009/crispri-induced-suppression-of-fimbriae-gene-fimh-of-a-uropathogenic-escherichia-coli-an-approach-to-inhibit-microbial-biofilms
#2
Azna Zuberi, Nayeem Ahmad, Asad U Khan
Urinary tract infection (UTI) is one the common infections caused by the recalcitrant nature of biofilms, developed after the pathogen has adhered to the inner lining of the urinary tract. Although significant research has been made in recent years to control these types of infection, but as of yet, no approach has sufficiently been able to reduce the prevalence of UTIs. The main objective of this study was to prevent UTIs through targeting the fimH gene, which is the major virulent factor responsible for biofilm formation...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29174524/engineering-cell-wall-synthesis-mechanism-for-enhanced-phb-accumulation-in-e-coli
#3
Xing-Chen Zhang, Yingying Guo, Xu Liu, Xin-Guang Chen, Qiong Wu, Guo-Qiang Chen
The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E...
November 24, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/29170951/bacterial-genome-editing-strategy-for-control-of-transcription-and-protein-stability
#4
Ida Lauritsen, Virginia Martínez, Carlotta Ronda, Alex Toftgaard Nielsen, Morten H H Nørholm
In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29158600/crispr-cas9-library-screening-for-drug-target-discovery
#5
REVIEW
Morito Kurata, Kouhei Yamamoto, Branden S Moriarity, Masanobu Kitagawa, David A Largaespada
CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms...
November 20, 2017: Journal of Human Genetics
https://www.readbyqxmd.com/read/29149479/cripsr-cas-expands-dynamic-range-of-gene-expression-from-t7rnap-promoters
#6
Sean R McCutcheon, Kwan Lun Chiu, Daniel D Lewis, Cheemeng Tan
BACKGROUND: Reducing leaky gene expression is critical for improving protein yield of recombinant bacteria and stability of engineered cellular circuits in synthetic biology. Leaky gene expression occurs when a genetic promoter is not fully repressed, leading to unintended protein synthesis in the absence of stimuli. Existing work has devised specific molecular strategies for reducing leaky gene expression of each promoter. Main Method and Results: In contrast, we describe a repurposed, modular CRISPRi system that attenuates leaky gene expression using a series of single-guide RNAs targeting the PT7/LacO1 ...
November 17, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/29112727/application-of-crispr-interference-for-metabolic-engineering-of-the-heterocyst-forming-multicellular-cyanobacterium-anabaena-sp-pcc-7120
#7
Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, Shigeki Ehira, Toru Hisabori
Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA (Higo A...
November 3, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/29105733/engineering-escherichia-coli-for-malate-production-by-integrating-modular-pathway-characterization-with-crispri-guided-multiplexed-metabolic-tuning
#8
Cong Gao, Shihui Wang, Guipeng Hu, Liang Guo, Xiulai Chen, Peng Xu, Liming Liu
The application of rational design in reallocating metabolic flux to overproduce desired chemicals is always restricted by the native regulatory network. Here, we demonstrated that in vitro modular pathway optimization combined with in vivo multiplexed combinatorial engineering enables effective characterization of the bottleneck of a complex biosynthetic cascade and improves the output of the engineered pathway. As a proof of concept, we systematically identified the rate-limiting step of a five-gene malate biosynthetic pathway by combinatorially tuning the enzyme loads of a reconstituted biocatalytic reaction in a cell-free system...
November 4, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29100516/crispr-interference-guided-multiplex-repression-of-endogenous-competing-pathway-genes-for-redirecting-metabolic-flux-in-escherichia-coli
#9
Seong Keun Kim, Wonjae Seong, Gui Hwan Han, Dae-Hee Lee, Seung-Goo Lee
BACKGROUND: Multiplex control of metabolic pathway genes is essential for maximizing product titers and conversion yields of fuels, chemicals, and pharmaceuticals in metabolic engineering. To achieve this goal, artificial transcriptional regulators, such as clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi), have been developed to specifically repress genes of interest. RESULTS: In this study, we deployed a tunable CRISPRi system for multiplex repression of competing pathway genes and, thus, directed carbon flux toward production of molecules of interest in Escherichia coli...
November 3, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29089930/transcriptomic-changes-in-response-to-putrescine-production-in-metabolically-engineered-corynebacterium-glutamicum
#10
Zhen Li, Jian-Zhong Liu
Putrescine is widely used in industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Although engineered Corynebacterium glutamicum has been successfully used to produce high levels of putrescine, the overall cellular physiological and metabolic changes caused by overproduction of putrescine remains unclear. To reveal the transcriptional changes that occur in response to putrescine production in an engineered C. glutamicum strain, a comparative transcriptomic analysis was carried out...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29082292/protocol-for-construction-of-a-tunable-crispr-interference-tcrispri-strain-for-escherichia-coli
#11
Xin-Tian Li, Cindy Sou, Suckjoon Jun
We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties...
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29059299/a-cautionary-tale-of-sense-antisense-gene-pairs-independent-regulation-despite-inverse-correlation-of-expression
#12
Ashish Goyal, Evgenij Fiškin, Tony Gutschner, Maria Polycarpou-Schwarz, Matthias Groß, Julia Neugebauer, Minakshi Gandhi, Maiwen Caudron-Herger, Vladimir Benes, Sven Diederichs
Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage...
October 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29058669/crispri-is-not-strand-specific-at-all-loci-and-redefines-the-transcriptional-landscape
#13
Françoise S Howe, Andrew Russell, Anna R Lamstaes, Afaf El-Sagheer, Anitha Nair, Tom Brown, Jane Mellor
CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts...
October 23, 2017: ELife
https://www.readbyqxmd.com/read/29051490/computational-design-of-small-transcription-activating-rnas-for-versatile-and-dynamic-gene-regulation
#14
James Chappell, Alexandra Westbrook, Matthew Verosloff, Julius B Lucks
A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design-allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to ~ 9000-fold gene activation...
October 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/29035510/crispri-and-crispra-screens-in-mammalian-cells-for-precision-biology-and-medicine
#15
Martin Kampmann
Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidating gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases...
October 24, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28985505/combined-crispri-a-based-chemical-genetic-screens-reveal-that-rigosertib-is-a-microtubule-destabilizing-agent
#16
Marco Jost, Yuwen Chen, Luke A Gilbert, Max A Horlbeck, Lenno Krenning, Grégory Menchon, Ankit Rai, Min Y Cho, Jacob J Stern, Andrea E Prota, Martin Kampmann, Anna Akhmanova, Michel O Steinmetz, Marvin E Tanenbaum, Jonathan S Weissman
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target...
October 5, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28981713/crispr-cas9-based-genome-editing-for-simultaneous-interference-with-gene-expression-and-protein-stability
#17
Virginia Martínez, Ida Lauritsen, Tonja Hobel, Songyuan Li, Alex Toftgaard Nielsen, Morten H H Nørholm
Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing protein levels. Here, we describe an efficient genome editing approach that affects specific protein abundances by changing the rates of both RNA synthesis and protein degradation, based on the two cross-kingdom control mechanisms CRISPRi and the N-end rule for protein stability...
November 16, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28941341/-application-of-genome-engineering-in-medical-synthetic-biology
#18
Fangyuan Wang, Dehua Zhao, Lei Stanley Qi
Synthetic biology aims to establish a complete set of engineering principles, theories, and methods, via the rational design and assembly of basic biological parts, for the goal of effective implementation of complex biological systems with programmable functions. In recent years, with emerging novel classes of programmable genetic parts, in particular, the establishment and optimization of CRISPR and CRISPRi technology platforms, synthetic biology is entering a new era. This review summarizes recent advances on CRISPR genome editing and gene regulation technologies, their applications in constructing programmable biological parts, and their roles in building sophisticated gene circuits...
March 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/28858701/systems-level-interference-strategies-to-decipher-host-factors-involved-in-bacterial-pathogen-interaction-from-rnai-to-crispri
#19
REVIEW
Maxime Québatte, Christoph Dehio
Bacterial pathogen-host cell interactions involve an intricate interplay of multiple components from both partners. Systems level surveys have been used widely to profile host requirements for pathogen infection. Functional genomics, and more specifically genome-wide perturbation screens, constitute attractive methodologies to assess such host infectomes. Although these strategies have successfully identified numerous critical host factors, they may have failed in generating the high-quality data required for systems level analysis...
September 1, 2017: Current Opinion in Microbiology
https://www.readbyqxmd.com/read/28854333/combining-crispr-and-crispri-systems-for-metabolic-engineering-of-e-%C3%A2-coli-and-1-4-bdo-biosynthesis
#20
Meng-Ying Wu, Li-Yu Sung, Hung Li, Chun-Hung Huang, Yu-Chen Hu
Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6...
September 12, 2017: ACS Synthetic Biology
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