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https://www.readbyqxmd.com/read/27846887/crispr-interference-crispri-for-gene-regulation-and-succinate-production-in-cyanobacterium-s-elongatus-pcc-7942
#1
Chun-Hung Huang, Claire R Shen, Hung Li, Li-Yu Sung, Meng-Ying Wu, Yu-Chen Hu
BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27842593/easy-regulation-of-metabolic-flux-in-escherichia-coli-using-an-endogenous-type-i-e-crispr-cas-system
#2
Yizhao Chang, Tianyuan Su, Qingsheng Qi, Quanfeng Liang
BACKGROUND: Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. RESULTS: By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27794593/artificial-symmetry-breaking-for-morphogenetic-engineering-bacterial-colonies
#3
Isaac N Nuñez, Tamara F Matute, Ilenne Del Valle, Anton Kan, Atri Choksi, Drew Endy, Jim Haseloff, Timothy Rudge, Fernan Federici
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794465/enhanced-solvent-production-by-metabolic-engineering-of-a-twin-clostridial-consortium
#4
Zhiqiang Wen, Nigel P Minton, Ying Zhang, Qi Li, Jinle Liu, Yu Jiang, Sheng Yang
The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production...
October 26, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27765793/a-precision-medicine-approach-to-the-rescue-of-function-on-malignant-calmodulinopathic-long-qt-syndrome
#5
Worawan B Limpitikul, Ivy E Dick, David Tester, Nicole J Boczek, Pattraranee Limphong, Wanjun Yang, Myoung Hyun Choi, Jennifer Babich, Deborah DiSilvestre, Ronald J Kanter, Gordon F Tomaselli, Michael J Ackerman, David Yue
RATIONALE: Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long QT syndrome (LQTS) with mutations involving either CALM1, CALM2, or CALM3 The underlying basis of this form of LQTS is a disruption of Ca(2+)/CaM-dependent inactivation (CDI) of L-type Ca(2+) channels (LTCCs). OBJECTIVE: To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS...
October 20, 2016: Circulation Research
https://www.readbyqxmd.com/read/27708057/systematic-mapping-of-functional-enhancer-promoter-connections-with-crispr-interference
#6
Charles P Fulco, Mathias Munschauer, Rockwell Anyoha, Glen Munson, Sharon R Grossman, Elizabeth M Perez, Michael Kane, Brian Cleary, Eric S Lander, Jesse M Engreitz
Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation...
November 11, 2016: Science
https://www.readbyqxmd.com/read/27693319/crispri-engineering-e-coli-for-morphology-diversification
#7
Dina Elhadi, Li Lv, Xiao-Ran Jiang, Hong Wu, Guo-Qiang Chen
Microbial morphology engineering has recently become interesting for biotechnology. Genes ftsZ and mreB encoding proteins of bacterial fission ring and skeletons, respectively, are essential for cell growth, they both are the most important genes keeping the bacterial shapes including the cell length and width, respectively. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used in this study to regulate expression intensities of ftsZ or/and mreB in E...
September 28, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27661255/compact-and-highly-active-next-generation-libraries-for-crispr-mediated-gene-repression-and-activation
#8
Max A Horlbeck, Luke A Gilbert, Jacqueline E Villalta, Britt Adamson, Ryan A Pak, Yuwen Chen, Alexander P Fields, Chong Yon Park, Jacob E Corn, Martin Kampmann, Jonathan S Weissman
We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes...
September 23, 2016: ELife
https://www.readbyqxmd.com/read/27647432/enhanced-protein-and-biochemical-production-using-crispri-based-growth-switches
#9
Songyuan Li, Christian Bille Jendresen, Alexander Grünberger, Carlotta Ronda, Sheila Ingemann Jensen, Stephan Noack, Alex Toftgaard Nielsen
Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2...
September 16, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27623797/application-of-crispri-in-corynebacterium-glutamicum-for-shikimic-acid-production
#10
Bo Zhang, Zhi-Qiang Liu, Chang Liu, Yu-Guo Zheng
OBJECTIVES: To construct, test and exploit the CRISPRi system for enhancement of shikimic acid production with Corynebacterium glutamicum. RESULTS: The CRISPRi system was used to regulate C. glutamicum gene expression at the transcriptional level. Hfq protein-mediated small regulatory RNAs system was compared with CRISPRi system. The more efficient CRISPRi system was used to adjust the metabolic flux involving the shikimic acid (SA) synthetic pathway. In 11 candidate genes, including transcription regulator, three targets were effective for increasing SA production...
September 13, 2016: Biotechnology Letters
https://www.readbyqxmd.com/read/27587996/the-development-of-a-viral-mediated-crispr-cas9-system-with-doxycycline-dependent-grna-expression-for-inducible-in-vitro-and-in-vivo-genome-editing
#11
Christopher A de Solis, Anthony Ho, Roopashri Holehonnur, Jonathan E Ploski
The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter...
2016: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/27569599/crispr-interference-guided-balancing-of-a-biosynthetic-mevalonate-pathway-increases-terpenoid-production
#12
Seong Keun Kim, Gui Hwan Han, Wonjae Seong, Haseong Kim, Seon-Won Kim, Dae-Hee Lee, Seung-Goo Lee
Methods for simple and efficient regulation of metabolic pathway genes are essential for maximizing product titers and conversion yields, and for minimizing the metabolic burden caused by heterologous expression of multiple genes often in the operon context. Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) is emerging as a promising tool for transcriptional modulation. In this study, we developed a regulatable CRISPRi system for fine-tuning biosynthetic pathways and thus directing carbon flux toward target product synthesis...
August 26, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27529184/biosensing-vibrio-cholerae-with-genetically-engineered-escherichia-coli
#13
Maciej B Holowko, Huijuan Wang, Premkumar Jayaraman, Chueh Loo Poh
Cholera is a potentially mortal, infectious disease caused by Vibrio cholerae bacterium. Current treatment methods of cholera still have limitations. Beneficial microbes that could sense and kill the V. cholerae could offer potential alternative to preventing and treating cholera. However, such V. cholerae targeting microbe is still not available. This microbe requires a sensing system to be able to detect the presence of V. cholera bacterium. To this end, we designed and created a synthetic genetic sensing system using nonpathogenic Escherichia coli as the host...
August 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27528508/depletion-of-undecaprenyl-pyrophosphate-phosphatases-disrupts-cell-envelope-biogenesis-in-bacillus-subtilis
#14
Heng Zhao, Yingjie Sun, Jason M Peters, Carol A Gross, Ethan C Garner, John D Helmann
: The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases...
November 1, 2016: Journal of Bacteriology
https://www.readbyqxmd.com/read/27498870/high-resolution-mapping-of-rna-polymerases-identifies-mechanisms-of-sensitivity-and-resistance-to-bet-inhibitors-in-t-8-21-aml
#15
Yue Zhao, Qi Liu, Pankaj Acharya, Kristy R Stengel, Quanhu Sheng, Xiaofan Zhou, Hojoong Kwak, Melissa A Fischer, James E Bradner, Stephen A Strickland, Sanjay R Mohan, Michael R Savona, Bryan J Venters, Ming-Ming Zhou, John T Lis, Scott W Hiebert
Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML...
August 16, 2016: Cell Reports
https://www.readbyqxmd.com/read/27450541/recognizing-and-engineering-digital-like-logic-gates-and-switches-in-gene-regulatory-networks
#16
Robert W Bradley, Martin Buck, Baojun Wang
A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour...
October 2016: Current Opinion in Microbiology
https://www.readbyqxmd.com/read/27353328/optimizing-sgrna-position-markedly-improves-the-efficiency-of-crispr-dcas9-mediated-transcriptional-repression
#17
Aliaksandra Radzisheuskaya, Daria Shlyueva, Iris Müller, Kristian Helin
CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully defined. In this study, we systematically assessed how sgRNA position affects the efficiency of CRISPRi in human cells. We analyzed 155 sgRNAs targeting 41 genes and found that CRISPRi efficiency relies heavily on the precise recruitment of the effector complex to the target gene transcription start site (TSS)...
October 14, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27341390/precision-modulation-of-neurodegenerative-disease-related-gene-expression-in-human-ipsc-derived-neurons
#18
Sabrina Mahalia Heman-Ackah, Andrew Roger Bassett, Matthew John Andrew Wood
The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27292569/n-cadherin-is-key-to-expression-of-the-nucleus-pulposus-cell-phenotype-under-selective-substrate-culture-conditions
#19
Priscilla Y Hwang, Liufang Jing, Jun Chen, Foon-Lian Lim, Ruhang Tang, Hyowon Choi, Kenneth M Cheung, Makarand V Risbud, Charles A Gersbach, Farshid Guilak, Victor Y Leung, Lori A Setton
Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27260361/development-of-a-crispr-cas9-tool-kit-for-comprehensive-engineering-of-bacillus-subtilis
#20
Adam W Westbrook, Murray Moo-Young, C Perry Chou
UNLABELLED: The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation...
August 15, 2016: Applied and Environmental Microbiology
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