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https://www.readbyqxmd.com/read/28640207/reverse-gyrase-functions-in-genome-integrity-maintenance-by-protecting-dna-breaks-in-vivo
#1
Wenyuan Han, Xu Feng, Qunxin She
Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation...
June 22, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28620688/controlling-microbial-phb-synthesis-via-crispri
#2
Dan Li, Li Lv, Jin-Chun Chen, Guo-Qiang Chen
Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively...
June 15, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28618222/de-novo-biosynthesis-of-glutarate-via-%C3%AE-keto-acid-carbon-chain-extension-and-decarboxylation-pathway-in-escherichia-coli
#3
Jian Wang, Yifei Wu, Xinxiao Sun, Qipeng Yuan, Yajun Yan
Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the l-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli...
June 23, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28616968/crispri-srna-transcriptional-translational-regulation-of-extracellular-electron-transfer-in-shewanella-oneidensis
#4
Yingxiu Cao, Xiaofei Li, Feng Li, Hao Song
Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i...
June 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28603699/crispr-interference-crispri-inhibition-of-luxs-gene-expression-in-e-coli-an-approach-to-inhibit-biofilm
#5
Azna Zuberi, Lama Misba, Asad U Khan
Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, E. coli biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28567487/synthetic-biology-of-polyhydroxyalkanoates-pha
#6
De-Chuan Meng, Guo-Qiang Chen
Microbial polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters which have been extensively studied using synthetic biology and metabolic engineering methods for improving production and for widening its diversity. Synthetic biology has allowed PHA to become composition controllable random copolymers, homopolymers, and block copolymers. Recent developments showed that it is possible to establish a microbial platform for producing not only random copolymers with controllable monomers and their ratios but also structurally defined homopolymers and block copolymers...
June 1, 2017: Advances in Biochemical Engineering/biotechnology
https://www.readbyqxmd.com/read/28534478/complementary-information-derived-from-crispr-cas9-mediated-gene-deletion-and-suppression
#7
Joseph Rosenbluh, Han Xu, William Harrington, Stanley Gill, Xiaoxing Wang, Francisca Vazquez, David E Root, Aviad Tsherniak, William C Hahn
CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes...
May 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28530708/engineering-rgb-color-vision-into-escherichia-coli
#8
Jesus Fernandez-Rodriguez, Felix Moser, Miryoung Song, Christopher A Voigt
Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.
July 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28501380/crispri-mediated-phosphoenolpyruvate-carboxylase-regulation-to-enhance-the-production-of-lipid-in-chlamydomonas-reinhardtii
#9
Pei-Hsun Kao, I-Son Ng
In this study, CRISPRi (clustered regularly interspaced short palindromic repeats interference) was used for the first time to regulate expression of exogenously supplied rfp gene as a proof-of-concept, and endogenous PEPC1 gene as a proof-of-function in Chlamydomonas reinhardtii. The efficiency of 94% and stability of 7 generations via CRISPRi mediated gene regulation in C. reinhardtii have been demonstrated by RFP. Gene PEPC1 encoding proteins are essential for controlling the carbon flux that enters the TCA cycle and plays a crucial role in carbon partitioning of substrates in competition with lipid synthesis...
May 4, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28490437/high-throughput-crispri-phenotyping-identifies-new-essential-genes-in-streptococcus-pneumoniae
#10
Xue Liu, Clement Gallay, Morten Kjos, Arnau Domenech, Jelle Slager, Sebastiaan P van Kessel, Kèvin Knoops, Robin A Sorg, Jing-Ren Zhang, Jan-Willem Veening
Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%)...
May 10, 2017: Molecular Systems Biology
https://www.readbyqxmd.com/read/28481362/genetic-interaction-mapping-in-mammalian-cells-using-crispr-interference
#11
Dan Du, Assen Roguev, David E Gordon, Meng Chen, Si-Han Chen, Michael Shales, John Paul Shen, Trey Ideker, Prashant Mali, Lei S Qi, Nevan J Krogan
We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation...
June 2017: Nature Methods
https://www.readbyqxmd.com/read/28431229/functional-enhancer-screening-in-single-cells
#12
Joris van Arensbergen, Bas van Steensel
In this issue of Molecular Cell, Xie et al. (2017) introduce Mosaic-seq, a powerful technology that combines CRISPRi and single-cell RNA-seq. This method enables the high-throughput assessment of contributions of enhancers to gene regulation.
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28418635/enhancing-protein-production-yield-from-chinese-hamster-ovary-cells-by-crispr-interference
#13
Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, Yu-Chen Hu
Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhfr and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells...
May 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28381263/engineering-halomonas-species-td01-for-enhanced-polyhydroxyalkanoates-synthesis-via-crispri
#14
Wei Tao, Li Lv, Guo-Qiang Chen
BACKGROUND: Clustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels...
April 6, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28375596/efficient-transcriptional-gene-repression-by-type-v-a-crispr-cpf1-from-eubacterium-eligens
#15
Seong Keun Kim, Haseong Kim, Woo-Chan Ahn, Kwang-Hyun Park, Eui-Jeon Woo, Dae-Hee Lee, Seung-Goo Lee
Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is an emerging technology for artificial gene regulation. Type II CRISPR-Cas endonuclease Cas9 is the most widely used protein for gene regulation with CRISPRi. Here, we present type V-A CRISPR-Cas endonuclease Cpf1-based CRISPRi. We constructed an l-rhamnose-inducible CRISPRi system with DNase-deactivated Cpf1 from Eubacterium eligens (EedCpf1) and compared its performance with catalytically deactivated Cas9 from Streptococcus pyogenes (SpdCas9)...
April 11, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28165460/programmable-transcriptional-repression-in-mycobacteria-using-an-orthogonal-crispr-interference-platform
#16
Jeremy M Rock, Forrest F Hopkins, Alejandro Chavez, Marieme Diallo, Michael R Chase, Elias R Gerrick, Justin R Pritchard, George M Church, Eric J Rubin, Christopher M Sassetti, Dirk Schnappinger, Sarah M Fortune
The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria...
February 6, 2017: Nature Microbiology
https://www.readbyqxmd.com/read/28095853/crispri-mediated-metabolic-engineering-of-e-coli-for-o-methylated-anthocyanin-production
#17
Brady F Cress, Quentin D Leitz, Daniel C Kim, Teresita D Amore, Jon Y Suzuki, Robert J Linhardt, Mattheos A G Koffas
BACKGROUND: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet)...
January 17, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/27984733/a-multiplexed-single-cell-crispr-screening-platform-enables-systematic-dissection-of-the-unfolded-protein-response
#18
Britt Adamson, Thomas M Norman, Marco Jost, Min Y Cho, James K Nuñez, Yuwen Chen, Jacqueline E Villalta, Luke A Gilbert, Max A Horlbeck, Marco Y Hein, Ryan A Pak, Andrew N Gray, Carol A Gross, Atray Dixit, Oren Parnas, Aviv Regev, Jonathan S Weissman
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis...
December 15, 2016: Cell
https://www.readbyqxmd.com/read/27983975/current-status-of-potential-applications-of-repurposed-cas9-for-structural-and-functional-genomics-of-plants
#19
Kunal Seth, Harish
Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique...
October 28, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27980086/crispri-based-genome-scale-identification-of-functional-long-noncoding-rna-loci-in-human-cells
#20
S John Liu, Max A Horlbeck, Seung Woo Cho, Harjus S Birk, Martina Malatesta, Daniel He, Frank J Attenello, Jacqueline E Villalta, Min Y Cho, Yuwen Chen, Mohammad A Mandegar, Michael P Olvera, Luke A Gilbert, Bruce R Conklin, Howard Y Chang, Jonathan S Weissman, Daniel A Lim
The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type...
January 6, 2017: Science
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