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https://www.readbyqxmd.com/read/29678175/gene-repression-via-multiplex-grna-strategy-in-y-lipolytica
#1
Jin-Lai Zhang, Yang-Zi Peng, Duo Liu, Hong Liu, Ying-Xiu Cao, Bing-Zhi Li, Chun Li, Ying-Jin Yuan
BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest...
April 20, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29622042/engineering-crispr-interference-system-in-klebsiella-pneumoniae-for-attenuating-lactic-acid-synthesis
#2
Jingxuan Wang, Peng Zhao, Ying Li, Lida Xu, Pingfang Tian
BACKGROUND: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid. RESULTS: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae...
April 5, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29621180/applications-of-crispr-cas-system-to-bacterial-metabolic-engineering
#3
REVIEW
Suhyung Cho, Jongoh Shin, Byung-Kwan Cho
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome...
April 5, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29608282/genetic-engineering-of-bee-gut-microbiome-bacteria-with-a-toolkit-for-modular-assembly-of-broad-host-range-plasmids
#4
Sean P Leonard, Jiri Perutka, J Elijah Powell, Peng Geng, Darby Richhart, Michelle Byrom, Shaunak Kar, Bryan W Davies, Andrew D Ellington, Nancy Moran, Jeffrey E Barrick
Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters and other coding sequences in newly isolated bacteria...
April 2, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29540185/linc00210-drives-wnt-%C3%AE-catenin-signaling-activation-and-liver-tumor-progression-through-ctnnbip1-dependent-manner
#5
Xiaomin Fu, Xiaoyan Zhu, Fujun Qin, Yong Zhang, Jizhen Lin, Yuechao Ding, Zihe Yang, Yiman Shang, Li Wang, Qinxian Zhang, Quanli Gao
BACKGROUND: Liver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown. METHODS: LncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH...
March 14, 2018: Molecular Cancer
https://www.readbyqxmd.com/read/29535977/feasibility-of-a-conditional-knockout-system-for-chlamydia-based-on-crispr-interference
#6
Scot P Ouellette
Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C...
2018: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/29519933/tuning-dcas9-s-ability-to-block-transcription-enables-robust-noiseless-knockdown-of-bacterial-genes
#7
Antoine Vigouroux, Enno Oldewurtel, Lun Cui, David Bikard, Sven van Teeffelen
Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts...
March 8, 2018: Molecular Systems Biology
https://www.readbyqxmd.com/read/29467311/epstein-barr-virus-nuclear-antigen-leader-protein-coactivates-ep300
#8
Chong Wang, Hufeng Zhou, Yong Xue, Jun Liang, Yohei Narita, Catherine Gerdt, Amy Y Zheng, Runsheng Jiang, Stephen Trudeau, Chih-Wen Peng, Benjamin E Gewurz, Bo Zhao
Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation...
May 1, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29446747/crispr-cas9-genome-editing-a-promising-tool-for-therapeutic-applications-of-induced-pluripotent-stem-cells
#9
Yanli Zhang, Danuta Sastre, Feng Wang
Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci...
February 14, 2018: Current Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29444854/bet-bromodomain-proteins-regulate-enhancer-function-during-adipogenesis
#10
Jonathan D Brown, Zachary B Feldman, Sean P Doherty, Jaime M Reyes, Peter B Rahl, Charles Y Lin, Quanhu Sheng, Qiong Duan, Alexander J Federation, Andrew L Kung, Saptarsi M Haldar, Richard A Young, Jorge Plutzky, James E Bradner
Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation...
February 14, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29403034/crispr-interference-based-specific-and-efficient-gene-inactivation-in-the-brain
#11
Yi Zheng, Wei Shen, Jian Zhang, Bo Yang, Yao-Nan Liu, Huihui Qi, Xia Yu, Si-Yao Lu, Yun Chen, Yu-Zhou Xu, Yun Li, Fred H Gage, Shuangli Mi, Jun Yao
CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency...
February 5, 2018: Nature Neuroscience
https://www.readbyqxmd.com/read/29385696/crispr-cas9-genetic-analysis-of-virus-host-interactions
#12
REVIEW
Makda Gebre, Jason L Nomburg, Benjamin E Gewurz
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus-host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR-Cas9 for the loss-of-function analysis of host dependency factors...
January 30, 2018: Viruses
https://www.readbyqxmd.com/read/29377933/tailor-made-gene-silencing-of-staphylococcus-aureus-clinical-isolates-by-crispr-interference
#13
Yusuke Sato'o, Junzo Hisatsune, Liansheng Yu, Tetsushi Sakuma, Takashi Yamamoto, Motoyuki Sugai
Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for Staphylococcus aureus...
2018: PloS One
https://www.readbyqxmd.com/read/29366319/molecular-toolkit-for-gene-expression-control-and-genome-modification-in-rhodococcus-opacus-pd630
#14
Drew M DeLorenzo, Austin G Rottinghaus, William R Henson, Tae Seok Moon
Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R...
February 16, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29359686/targeting-ras-driven-human-cancer-cells-with-antibodies-to-upregulated-and-essential-cell-surface-proteins
#15
Alexander J Martinko, Charles Truillet, Olivier Julien, Juan E Diaz, Max A Horlbeck, Gordon Whiteley, Josip Blonder, Jonathan S Weissman, Sourav Bandyopadhyay, Michael J Evans, James A Wells
While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations...
January 23, 2018: ELife
https://www.readbyqxmd.com/read/29344811/metabolic-evolution-and-a-comparative-omics-analysis-of-corynebacterium-glutamicum-for-putrescine-production
#16
Zhen Li, Yu-Ping Shen, Xuan-Long Jiang, Li-Shen Feng, Jian-Zhong Liu
Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum...
January 17, 2018: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/29343791/single-plasmid-systems-for-inducible-dual-protein-expression-and-for-crispr-cas9-crispri-gene-regulation-in-lactic-acid-bacterium-lactococcus-lactis
#17
Aleš Berlec, Katja Škrlec, Janja Kocjan, Maria Olenic, Borut Štrukelj
Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed...
January 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29337370/modulating-gene-expression-in-epstein-barr-virus-ebv-positive-b-cell-lines-with-crispra-and-crispri
#18
Liang Wei Wang, Stephen J Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29316926/rna-guided-single-double-gene-repressions-in-corynebacterium-glutamicum-using-an-efficient-crispr-interference-and-its-application-to-industrial-strain
#19
Jaehyun Park, Hyojung Shin, Sun-Mi Lee, Youngsoon Um, Han Min Woo
BACKGROUND: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host. RESULTS: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously...
January 9, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29311279/a-robust-crispr-interference-gene-repression-system-in-pseudomonas
#20
Sue Zanne Tan, Christopher R Reisch, Kristala L J Prather
Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida This inducible system enables the study of essential genes, as shown by ftsZ depletions in P...
April 1, 2018: Journal of Bacteriology
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