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https://www.readbyqxmd.com/read/29329593/targeting-ectodysplasin-promotor-by-crispr-dcas9-effector-effectively-induces-the-reprogramming-of-human-bone-marrow-derived-mesenchymal-stem-cells-into-sweat-gland-like-cells
#1
Sujing Sun, Jun Xiao, Jiahui Huo, Zhijun Geng, Kui Ma, Xiaoyan Sun, Xiaobing Fu
BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs)...
January 12, 2018: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29311279/a-robust-crispri-gene-repression-system-in-pseudomonas
#2
Sue Zanne Tan, Christopher R Reisch, Kristala L J Prather
Pseudomonas spp. are widely used model organisms in different areas of research. Despite its relevance in many applications, protein depletion tools in this host remain limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using the Streptococcus pasteurianus 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 P. putida This inducible system enables the study of essential genes, as shown by ftsZ depletions in P...
January 8, 2018: Journal of Bacteriology
https://www.readbyqxmd.com/read/29286403/a-universal-protocol-for-large-scale-grna-library-production-from-any-dna-source
#3
Anna Köferle, Stefan H Stricker
The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment...
December 6, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29259522/applications-of-crispr-cas9-in-the-mammalian-central-nervous-system%C3%A2-%C3%A2
#4
REVIEW
Katherine E Savell, Jeremy J Day
Within the central nervous system, gene regulatory mechanisms are crucial regulators of cellular development and function, and dysregulation of these systems is commonly observed in major neuropsychiatric and neurological disorders. However, due to a lack of tools to specifically modulate the genome and epigenome in the central nervous system, many molecular and genetic mechanisms underlying cognitive function and behavior are still unknown. Although genome editing tools have been around for decades, the recent emergence of inexpensive, straightforward, and widely accessible CRISPR/Cas9 systems has led to a revolution in gene editing...
December 2017: Yale Journal of Biology and Medicine
https://www.readbyqxmd.com/read/29203910/tet-proteins-safeguard-bivalent-promoters-from-de-novo-methylation-in-human-embryonic-stem-cells
#5
Nipun Verma, Heng Pan, Louis C Doré, Abhijit Shukla, Qing V Li, Bobbie Pelham-Webb, Virginia Teijeiro, Federico González, Andrei Krivtsov, Chan-Jung Chang, Eirini P Papapetrou, Chuan He, Olivier Elemento, Danwei Huangfu
TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state...
January 2018: Nature Genetics
https://www.readbyqxmd.com/read/29197638/robust-transcriptional-activation-in-plants-using-multiplexed-crispr-act2-0-and-mtale-act-systems
#6
Levi G Lowder, Jianping Zhou, Yingxiao Zhang, Aimee Malzahn, Zhaohui Zhong, Tzung-Fu Hsieh, Daniel F Voytas, Yong Zhang, Yiping Qi
User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR-associated protein 9 (dCas9) fused with four tandem repeats of the transcriptional activator VP16 (VP64) could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCas9-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2...
November 29, 2017: Molecular Plant
https://www.readbyqxmd.com/read/29197619/harnessing-crispr-cas-systems-for-programmable-transcriptional-and-post-transcriptional-regulation
#7
REVIEW
Ahmed Mahas, C Neal Stewart, Magdy M Mahfouz
Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications...
November 29, 2017: Biotechnology Advances
https://www.readbyqxmd.com/read/29185024/sirt1-overexpression-suppresses-fluoride-induced-p53-acetylation-to-alleviate-fluoride-toxicity-in-ameloblasts-responsible-for-enamel-formation
#8
Maiko Suzuki, Atsushi Ikeda, John D Bartlett
Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo...
November 28, 2017: Archives of Toxicology
https://www.readbyqxmd.com/read/29173171/new-developments-in-crispr-cas-based-functional-genomics-and-their-implications-for-research-using-zebrafish
#9
Sergey V Prykhozhij, Lucia Caceres, Jason N Berman
Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish...
November 21, 2017: Current Gene Therapy
https://www.readbyqxmd.com/read/29171881/rna-n6-methyladenosine-methyltransferase-mettl3-promotes-liver-cancer-progression-through-ythdf2-dependent-post-transcriptional-silencing-of-socs2
#10
Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice Ho-Ching Tsang, Jialing Shen, Carol Lai-Hung Cheng, Long-Hin Tsang, Daniel Wai-Hung Ho, David Kung-Chun Chiu, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Chun-Ming Wong
Epigenetic alterations immensely contributed to human carcinogenesis. Conventional epigenetic studies predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications on RNAs emerge as a new layer of epigenetic regulation. N6-methyladenosine (m6A) is the most abundant chemical modification on eukaryotic mRNA and is important to the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that METTL3 (methyltransferase like 3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors...
November 24, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29158545/a-potent-cas9-derived-gene-activator-for-plant-and-mammalian-cells
#11
Zhenxiang Li, Dandan Zhang, Xiangyu Xiong, Bingyu Yan, Wei Xie, Jen Sheen, Jian-Feng Li
Overexpression of complementary DNA represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labour for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at the endogenous genomic locus through a programmable DNA-binding module...
November 20, 2017: Nature Plants
https://www.readbyqxmd.com/read/29158476/programmable-dna-looping-using-engineered-bivalent-dcas9-complexes
#12
Nan Hao, Keith E Shearwin, Ian B Dodd
DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E...
November 20, 2017: Nature Communications
https://www.readbyqxmd.com/read/29133799/hit-and-run-epigenetic-editing-prevents-senescence-entry-in-primary-breast-cells-from-healthy-donors
#13
Emily A Saunderson, Peter Stepper, Jennifer J Gomm, Lily Hoa, Adrienne Morgan, Michael D Allen, J Louise Jones, John G Gribben, Tomasz P Jurkowski, Gabriella Ficz
Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest...
November 13, 2017: Nature Communications
https://www.readbyqxmd.com/read/29126880/a-heart-enriched-antisense-long-non-coding-rna-regulates-the-balance-between-cardiac-and-skeletal-muscle-triadin
#14
Lu Zhang, Antonio Salgado-Somoza, Melanie Vausort, Przemyslaw Leszek, Yvan Devaux
Non-coding RNAs play major roles in cardiac pathophysiology. Recent studies reported that long non-coding RNAs (lncRNAs) are dysregulated in the failing heart, but how they contribute to heart failure development is unclear. In this study, we aimed to identify heart-enriched lncRNAs and investigate their regulation and function in the failing heart. RESULTS: Analysis of a RNA-seq dataset of 15 Caucasian tissues allowed the identification of 415 heart-enriched lncRNAs. Fifty-three lncRNAs were located on the genome in close vicinity to protein-coding genes associated with cardiac function and disease...
November 8, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29125739/yield-improvement-of-the-anti-mrsa-antibiotics-wap-8294a-by-crispr-dcas9-combined-with-refactoring-self-protection-genes-in-lysobacter-enzymogenes-oh11
#15
Lingjun Yu, Wei Su, Paul D Fey, Fengquan Liu, Liangcheng Du
The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production...
November 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29112727/application-of-crispr-interference-for-metabolic-engineering-of-the-heterocyst-forming-multicellular-cyanobacterium-anabaena-sp-pcc-7120
#16
Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, Shigeki Ehira, Toru Hisabori
Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA (Higo A...
November 3, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/29103811/crispr-cas9-based-plant-genome-editing-significance-opportunities-and-recent-advances
#17
Neelam Soda, Lokesh Verma, Jitender Giri
Precise genome editing is a quantum leap in the field of plant sciences. Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas9 protein have emerged as a powerful tool for precise genome editing. CRISPR-Cas9 system introduces small heritable mutations (indels) in the genome of an organism. This system also enables precise gene characterization in plants with complex genomes. Besides, it offers new opportunities of trait stacking, where addition of desirable traits or removal of undesirable traits can be achieved simultaneously in a single event...
October 27, 2017: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/29091290/combinatorial-genetics-in-liver-repopulation-and-carcinogenesis-with-a-novel-in-vivo-crispr-activation-platform
#18
Kirk J Wangensteen, Yue J Wang, Zhixun Dou, Amber W Wang, Elham Mosleh-Shirazi, Max A Horlbeck, Luke A Gilbert, Jonathan S Weissman, Shelley L Berger, Klaus H Kaestner
CRISPR/Cas9 activation (CRISPRa) systems have enabled genetic screens in cultured cells lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the dCas9(+) mouse, a Cre-inducible CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation...
November 1, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29080263/genomes-in-focus-development-and-applications-of-crispr-cas9-imaging-technologies
#19
Spencer Knight, Robert Tjian, Jennifer Doudna
The discovery of the CRISPR-Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA-guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genomic imaging. This review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer new mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo...
October 27, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/29077398/control-of-adipogenic-differentiation-in-mesenchymal-stem-cells-via-endogenous-gene-activation-using-crispr-cas9
#20
Yuichi Furuhata, Yuta Nihongaki, Moritoshi Sato, Keitaro Yoshimoto
Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the in vivo differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue-specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system and achieved efficient induction of different types of adipocyte-like cells from MSCs...
November 7, 2017: ACS Synthetic Biology
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