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https://www.readbyqxmd.com/read/28623586/multiplexed-transcriptional-activation-or-repression-in-plants-using-crispr-dcas9-based-systems
#1
Levi G Lowder, Joseph W Paul, Yiping Qi
Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28623229/zebrafish-znfl1s-control-the-expression-of-hoxb1b-in-the-posterior-neuroectoderm-by-acting-upstream-of-pou5f3-and-sall4
#2
Xiaohua Dong, Jingyun Li, Luqingqing He, Chun Gu, Wenshuang Jia, Yunyun Yue, Jun Li, Qinxin Zhang, Lele Chu, Qingshun Zhao
Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to express in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in zebrafish genome and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down by using morpholino to inhibit their translations or dCas9-Eve to inhibit their transcriptions, the zebrafish gastrula displays reduced expression of hoxb1b, the maker gene for the posterior neuroectoderm...
June 16, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28622347/rna-guided-transcriptional-activation-via-crispr-dcas9-mimics-overexpression-phenotypes-in-arabidopsis
#3
Jong-Jin Park, Emma Dempewolf, Wenzheng Zhang, Zeng-Yu Wang
Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants...
2017: PloS One
https://www.readbyqxmd.com/read/28607761/multiplex-gene-regulation-by-crispr-ddcpf1
#4
Xiaochun Zhang, Jingman Wang, Qiuxiang Cheng, Xuan Zheng, Guoping Zhao, Jin Wang
The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9...
2017: Cell Discovery
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/28557624/rna-guided-activation-of-pluripotency-genes-in-human-fibroblasts
#6
Kai Xiong, Yan Zhou, Kristian Aabo Blichfeld, Poul Hyttel, Lars Bolund, Kristine Karla Freude, Yonglun Luo
Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts)...
June 2017: Cellular Reprogramming
https://www.readbyqxmd.com/read/28547585/aio-casilio-a-robust-crispr-cas9-pumilio-system-for-chromosome-labeling
#7
Shuxian Zhang, Zhan Song
The biological function of chromatin bases on its spatial organization and dynamic activities in different situations. Labeling and tracing of genomic sequences has been a huge challenge in studying the spatial dynamics of chromatin. We reported the development of 'all-in-one Casilio system (Aio-Casilio)', a new system that enables the labeling of endogenous genomic loci. The Aio-Casilio system consists of the dCas9 protein, mClover fused with the Pumilio and FBF proteins RNA-binding domain (PUF domain) and an U6-sgRNA appended with multiple PUF-binding site(s)...
May 25, 2017: Journal of Molecular Histology
https://www.readbyqxmd.com/read/28542388/targeted-dna-methylation-in-pericentromeres-with-genome-editing-based-artificial-dna-methyltransferase
#8
Taiga Yamazaki, Yu Hatano, Tetsuya Handa, Sakiko Kato, Kensuke Hoida, Rui Yamamura, Takashi Fukuyama, Takayuki Uematsu, Noritada Kobayashi, Hiroshi Kimura, Kazuo Yamagata
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI...
2017: PloS One
https://www.readbyqxmd.com/read/28541304/digital-logic-circuits-in-yeast-with-crispr-dcas9-nor-gates
#9
Miles W Gander, Justin D Vrana, William E Voje, James M Carothers, Eric Klavins
Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses...
May 25, 2017: Nature Communications
https://www.readbyqxmd.com/read/28534478/complementary-information-derived-from-crispr-cas9-mediated-gene-deletion-and-suppression
#10
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/28522389/crispr-dcas9-mediated-inhibition-of-gene-expression-in-staphylococcus-aureus
#11
Xiaoyun Dong, Yingli Jin, Di Ming, Bangbang Li, Haisi Dong, Lin Wang, Tiedong Wang, Dacheng Wang
The understanding of the genetic mechanism of Staphylococcus aureus requires efficient tools, however, genetic manipulation in S. aureus is always laborious and time-consuming. Here we proposed a novel CRISPR/dCas9 interference method for the rapid knockdown of target genes. Furthermore, multiple genes can be repressed simultaneously by using this method.
May 15, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28509419/live-cell-crispr-imaging-in-plants-reveals-dynamic-telomere-movements
#12
Steven Dreissig, Simon Schiml, Patrick Schindele, Oda Weiss, Twan Rutten, Veit Schubert, Evgeny Gladilin, Michael Florian Mette, Holger Puchta, Andreas Houben
Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system...
May 16, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28503202/stabilization-of-foxp3-expression-by-crispr-dcas9-based-epigenome-editing-in-mouse-primary-t-cells
#13
Masahiro Okada, Mitsuhiro Kanamori, Kazue Someya, Hiroko Nakatsukasa, Akihiko Yoshimura
BACKGROUND: Epigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types. For functional epigenome editing, assessing the chromatin context-dependent activity of artificial epigenetic modifier is required. RESULTS: In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to mouse primary T cells, focusing on the Forkhead box P3 (Foxp3) gene locus, a master transcription factor of regulatory T cells (Tregs)...
2017: Epigenetics & Chromatin
https://www.readbyqxmd.com/read/28495970/high-throughput-biochemical-profiling-reveals-sequence-determinants-of-dcas9-off-target-binding-and-unbinding
#14
Evan A Boyle, Johan O L Andreasson, Lauren M Chircus, Samuel H Sternberg, Michelle J Wu, Chantal K Guegler, Jennifer A Doudna, William J Greenleaf
The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM)...
May 23, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28451358/synthetically-controlling-dendrimer-flexibility-improves-delivery-of-large-plasmid-dna
#15
Jessica A Kretzmann, Diwei Ho, Cameron W Evans, Janice H C Plani-Lam, Benjamin Garcia-Bloj, A Elaaf Mohamed, Megan L O'Mara, Ethan Ford, Dennis E K Tan, Ryan Lister, Pilar Blancafort, Marck Norret, K Swaminathan Iyer
Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity...
April 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28448738/site-specific-recruitment-of-epigenetic-factors-with-a-modular-crispr-cas-system
#16
Tobias Anton, Sebastian Bultmann
Dissecting the complex network of epigenetic modifications requires tools that combine precise recognition of DNA sequences with the capability to modify epigenetic marks. The CRISPR/Cas system has been proven to be a valuable addition to existing methodologies that fulfill these tasks. So far, sequence-specific editing of epigenetic modifications such as DNA methylation and histone posttranslational modifications relied on direct fusions of enzymatically inactivated Cas9 (dCas9) with epigenetic effectors. Here, we report a novel, modular system that facilitates the recruitment of any GFP-tagged protein to desired genomic loci...
February 23, 2017: Nucleus
https://www.readbyqxmd.com/read/28446681/minute-virus-of-mice-mvm-inhibits-transcription-of-the-cyclin-b1-gene-during-infection
#17
Matthew S Fuller, Kinjal Majumder, David J Pintel
Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent pre-mitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others previously reported, depends upon the significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function thus preventing mitotic entry...
April 26, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28418635/enhancing-protein-production-yield-from-chinese-hamster-ovary-cells-by-crispr-interference
#18
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/28416141/multiplexed-engineering-and-analysis-of-combinatorial-enhancer-activity-in-single-cells
#19
Shiqi Xie, Jialei Duan, Boxun Li, Pei Zhou, Gary C Hon
The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28412663/a-facile-rapid-and-sensitive-detection-of-mrsa-using-a-crispr-mediated-dna-fish-method-antibody-like-dcas9-sgrna-complex
#20
Kyeonghye Guk, Joo Oak Keem, Seul Gee Hwang, Hyeran Kim, Taejoon Kang, Eun-Kyung Lim, Juyeon Jung
Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe...
September 15, 2017: Biosensors & Bioelectronics
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