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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
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 impact physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses CRISPR interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase (Mb) of sequence in the vicinity of 2 essential transcription factors, MYC and GATA1, and identify 9 distal enhancers that control gene expression and cellular proliferation...
September 29, 2016: Science
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
Max A Horlbeck, Luke A Gilbert, Jacqueline E Villalta, Britt Adamson, Ryan A Pak, Yuwen Chen, Alexander P Fields, Chong Y 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
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
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
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
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
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
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
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
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
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)...
June 28, 2016: Nucleic Acids Research
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
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
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
Qi Li, Jun Chen, Nigel P Minton, Ying Zhang, Zhiqiang Wen, Jinle Liu, Haifeng Yang, Zhe Zeng, Xiaodan Ren, Junjie Yang, Yang Gu, Weihong Jiang, Yu Jiang, Sheng Yang
Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6...
July 2016: Biotechnology Journal
Eira Choudhary, Ajitesh Lunge, Nisheeth Agarwal
Tremendous amount of physiological and functional complexities acquired through decades of evolutionary pressure makes Mycobacterium tuberculosis (Mtb) one of the most dreadful microorganisms infecting humans from centuries. Astonishing advances in genomics and genome editing tools substantially grew our knowledge about Mtb as an organism but dramatically failed to completely understand it as a pathogen. Though conventional tools based on homologous recombination, antisense, controlled proteolysis, etc. have made important contributions in advancing our understanding of the pathophysiology of Mtb, yet these approaches have not accentuated our exploration of mycobacterium on account of certain technical limitations...
May 2016: Tuberculosis
Volker F Wendisch, João M P Jorge, Fernando Pérez-García, Elvira Sgobba
L-Amino acids find various applications in biotechnology. L-Glutamic acid and its salts are used as flavor enhancers. Other L-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. L-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches...
June 2016: World Journal of Microbiology & Biotechnology
Jing-Long Liang, Li-Qiong Guo, Jun-Fang Lin, Ze-Qi He, Fa-Ji Cai, Jun-Fei Chen
Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli...
June 2016: World Journal of Microbiology & Biotechnology
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