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https://www.readbyqxmd.com/read/28985505/combined-crispri-a-based-chemical-genetic-screens-reveal-that-rigosertib-is-a-microtubule-destabilizing-agent
#1
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
#2
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...
September 7, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28941341/-application-of-genome-engineering-in-medical-synthetic-biology
#3
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
#4
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...
August 28, 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
#5
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
https://www.readbyqxmd.com/read/28832943/crispri-repression-of-nonhomologous-end-joining-for-enhanced-genome-engineering-via-homologous-recombination-in-yarrowia-lipolytica
#6
Cory Schwartz, Keith Frogue, Adithya Ramesh, Joshua Misa, Ian Wheeldon
In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica...
August 19, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28827812/crosstalk-between-the-tricarboxylic-acid-cycle-and-peptidoglycan-synthesis-in-caulobacter-crescentus-through-the-homeostatic-control-of-%C3%AE-ketoglutarate
#7
Irnov Irnov, Zhe Wang, Nicholas D Jannetty, Julian A Bustamante, Kyu Y Rhee, Christine Jacobs-Wagner
To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis...
August 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28732459/crispulator-a-discrete-simulation-tool-for-pooled-genetic-screens
#8
Tamas Nagy, Martin Kampmann
BACKGROUND: The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on the selection of optimal screen design parameters, which also affects cost and scalability. However, the cost and effort of implementing pooled screens prohibits experimental testing of a large number of parameters. RESULTS: We present CRISPulator, a Monte Carlo method-based computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy...
July 21, 2017: BMC Bioinformatics
https://www.readbyqxmd.com/read/28640207/reverse-gyrase-functions-in-genome-integrity-maintenance-by-protecting-dna-breaks-in-vivo
#9
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
#10
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
#11
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
#12
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...
September 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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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