scholarly journals Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

2020 ◽  
Vol 132 (23) ◽  
pp. 9083-9088 ◽  
Author(s):  
Wenyuan Zhou ◽  
Wes Brown ◽  
Anirban Bardhan ◽  
Michael Delaney ◽  
Amber S. Ilk ◽  
...  
Keyword(s):  
Guide Rna ◽  
Spatiotemporal Control ◽  
In Cells

scholarly journals Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

2019 ◽  
Author(s):  
Wenyuan Zhou ◽  
Wes Brown ◽  
Anirban Bardhan ◽  
Michael Delaney ◽  
Amber S. Ilk ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Zebrafish Embryos ◽  
Complete Suppression ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Conditional Control ◽  
Rapid Restoration ◽  
Spatiotemporal Control ◽  
In Cells

AbstractWe developed a new method for conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos via photochemically activated, caged guide RNAs. Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5’-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:target dsDNA hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off to on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. caged gRNAs are novel tools for conditional control of gene editing thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

scholarly journals Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

2020 ◽  
Vol 59 (23) ◽  
pp. 8998-9003 ◽  
Author(s):  
Wenyuan Zhou ◽  
Wes Brown ◽  
Anirban Bardhan ◽  
Michael Delaney ◽  
Amber S. Ilk ◽  
...  
Keyword(s):  
Guide Rna ◽  
Spatiotemporal Control ◽  
In Cells

Enzymatic noncovalent synthesis of peptide assemblies generates multimolecular crowding in cells for biomedical applications

2021 ◽  
Author(s):  
Meihui Yi ◽  
Weiyi Tan ◽  
Jiaqi Guo ◽  
Bing Xu
Keyword(s):  
Biomedical Applications ◽  
Building Blocks ◽  
Cellular Functions ◽  
Molecular Building Blocks ◽  
Spatiotemporal Control ◽  
In Cells

Enzymatic noncovalent synthesis enables spatiotemporal control of multimolecular crowding in cells, thus offering a unique opportunity for modulating cellular functions. This article introduces some representative enzymes and molecular building blocks...

scholarly journals Identification of a New Site of Sumoylation on Tel (ETV6) Uncovers a PIAS-Dependent Mode of Regulating Tel Function

2008 ◽  
Vol 28 (7) ◽  
pp. 2342-2357 ◽  
Author(s):  
M. Guy Roukens ◽  
Mariam Alloul-Ramdhani ◽  
Alfred C. O. Vertegaal ◽  
Zeinab Anvarian ◽  
Crina I. A. Balog ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Control Of Gene Expression ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Spatiotemporal Control ◽  
Identification And Characterization ◽  
In Cells

ABSTRACT Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.

scholarly journals Chemical control of a CRISPR-Cas9 acetyltransferase

2017 ◽  
Author(s):  
Jonathan H. Shrimp ◽  
Carissa Grose ◽  
Stephanie R. T. Widmeyer ◽  
Ajit Jadhav ◽  
Jordan L. Meier
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Critical Role ◽  
Regulation Of Transcription ◽  
Transcriptional Activators ◽  
Guide Rna ◽  
Transcriptional Reporter ◽  
Expression Control ◽  
Gene Expression Control ◽  
Spatiotemporal Control

AbstractLysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Towards this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators, and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype, and reveal a role for p300’s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tuneable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role, and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

1996 ◽  
Vol 54 ◽  
pp. 758-759
Author(s):  
D. W. Fairbain ◽  
M.D. Standing ◽  
K.L. O'Neill
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Resistant Cell ◽  
Membrane Blebbing ◽  
Late Loss ◽  
Induced Apoptosis ◽  
Dying Cells ◽  
In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

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