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https://www.readbyqxmd.com/read/29133799/hit-and-run-epigenetic-editing-prevents-senescence-entry-in-primary-breast-cells-from-healthy-donors
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
https://www.readbyqxmd.com/read/29059299/a-cautionary-tale-of-sense-antisense-gene-pairs-independent-regulation-despite-inverse-correlation-of-expression
#9
Ashish Goyal, Evgenij Fiškin, Tony Gutschner, Maria Polycarpou-Schwarz, Matthias Groß, Julia Neugebauer, Minakshi Gandhi, Maiwen Caudron-Herger, Vladimir Benes, Sven Diederichs
Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage...
October 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29058669/crispri-is-not-strand-specific-at-all-loci-and-redefines-the-transcriptional-landscape
#10
Françoise S Howe, Andrew Russell, Anna R Lamstaes, Afaf El-Sagheer, Anitha Nair, Tom Brown, Jane Mellor
CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts...
October 23, 2017: ELife
https://www.readbyqxmd.com/read/29056323/impeding-transcription-of-expanded-microsatellite-repeats-by-deactivated-cas9
#11
Belinda S Pinto, Tanvi Saxena, Ruan Oliveira, Héctor R Méndez-Gómez, John D Cleary, Lance T Denes, Ona McConnell, Juan Arboleda, Guangbin Xia, Maurice S Swanson, Eric T Wang
Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective...
November 2, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29053968/multiscale-3d-genome-rewiring-during-mouse-neural-development
#12
Boyan Bonev, Netta Mendelson Cohen, Quentin Szabo, Lauriane Fritsch, Giorgio L Papadopoulos, Yaniv Lubling, Xiaole Xu, Xiaodan Lv, Jean-Philippe Hugnot, Amos Tanay, Giacomo Cavalli
Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo...
October 19, 2017: Cell
https://www.readbyqxmd.com/read/29042431/in-situ-genotyping-of-a-pooled-strain-library-after-characterizing-complex-phenotypes
#13
Michael J Lawson, Daniel Camsund, Jimmy Larsson, Özden Baltekin, David Fange, Johan Elf
In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference...
October 17, 2017: Molecular Systems Biology
https://www.readbyqxmd.com/read/29038154/deactivated-crispr-associated-protein-9-for-minor-allele-enrichment-in-cell-free-dna
#14
Amin Aalipour, Jonathan C Dudley, Seung-Min Park, Surya Murty, Jacob J Chabon, Evan A Boyle, Maximilian Diehn, Sanjiv S Gambhir
BACKGROUND: Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations...
October 16, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/29031730/bidirectional-manipulation-of-gene-expression-in-adipocytes-using-crispra-and-sirna
#15
Morten Lundh, Kaja Pluciñska, Marie S Isidor, Patricia S S Petersen, Brice Emanuelli
OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations...
October 2017: Molecular Metabolism
https://www.readbyqxmd.com/read/29026112/engineering-species-like-barriers-to-sexual-reproduction
#16
Maciej Maselko, Stephen C Heinsch, Jeremy M Chacón, William R Harcombe, Michael J Smanski
Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator...
October 12, 2017: Nature Communications
https://www.readbyqxmd.com/read/28986912/plant-gene-regulation-using-multiplex-crispr-dcas9-artificial-transcription-factors
#17
Levi G Lowder, Aimee Malzahn, Yiping Qi
Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28979918/dcas9-mediated-transcriptional-activation-of-tissue-inhibitor-of-metalloproteinases
#18
Tyler Duellman, Andrea Doll, Xi Chen, Rie Wakamiya, Jay Yang
Selective gene activation with the dCas9 (deactivated clustered regularly interspaced short palindromic repeats [CRISPR] associated protein 9)/CRISPR targeting of a transcriptional activator effector is now well established. However, the optimal targeting of guide RNA (gRNA) for a given gene is largely a matter of trial and error. We explored the optimal targeting site for tissue inhibitor of metalloproteinases (TIMPs) by first screening multiple gRNA target sites using a luciferase-based promoter-reporter system and next confirmed the effective TIMP induction in the mouse motor neuron-like neuron-enriched spinal cord 34 (NSC34) cells...
2017: Metalloproteinases in Medicine
https://www.readbyqxmd.com/read/28973434/dcas9-based-epigenome-editing-suggests-acquisition-of-histone-methylation-is-not-sufficient-for-target-gene-repression
#19
Henriette O'Geen, Chonghua Ren, Charles M Nicolet, Andrew A Perez, Julian Halmai, Victoria M Le, Joel P Mackay, Peggy J Farnham, David J Segal
Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type...
September 29, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28963258/kinetics-of-dcas9-target-search-in-escherichia-coli
#20
Daniel Lawson Jones, Prune Leroy, Cecilia Unoson, David Fange, Vladimir Ćurić, Michael J Lawson, Johan Elf
How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays...
September 29, 2017: Science
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