89 research outputs found
Regulation of E(spl) Gene Expression During Development
The Notch pathway, a crucial developmental signaling system, acts to direct the fates of individual cells in many organisms and has also been implicated in a wide range of human diseases. Notch signaling plays a vital role in cell fate decisions in almost every tissue type ranging from the skin to the nervous and vascular systems. Aberrant Notch signaling has been implicated as a cause of many diseases, including a variety of cancers. Activation of the Notch receptor releases a Notch intracellular domain into the nucleus, where it binds with a transcription factor, Suppressor of Hairless (Su(H))to create an active complex which upregulates expression of target genes. In Drosophila the primary targets of Notch activation are the Enhancer of Split [E(spl)] genes. The E(spl) genes encode a family of basic-helix-loop-helix (bHLH) transcription factors, which exhibit overlapping functions throughout developmental stages. In order to determine the mechanisms through which E(spl) gene expression is controlled, I used three approaches to study E(spl) regulation. First, Bioinformatics analysis of the upstream regulatory regions of the E(spl) genes reveals binding sites for transcription factors that may act to regulate E(spl) gene expression. Evolutionary conservation of sites in the regulatory region lends support to their importance in the regulation of gene expression. Second, Real Time PCR quantification of the expression of three E(spl) genes at different stages of Drosophila metamorphosis suggest roles for some of these genes. Third, a reporter vector with the upstream region of one of the E(spl) genes cloned upstream of the firefly luciferase gene was constructed and used in Drosophila tissue culture experiments to further analyze the regulation of gene expression. Results from these three approaches will help to better understand the process of gene regulation and to characterize the mechanisms involved in controlling gene expression. Specific understanding of Notch target genes will elucidate how the Notch pathway functions in both normal and disease cells
CRISPR RNA-guided activation of endogenous human genes
Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells
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High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
CRISPR RNA-guided endonucleases (RGENs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications
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Robust, synergistic regulation of human gene expression using TALE activators
Artificial transcription activator-like (TAL) effector-based activators (TALE activators) have broad utility but previous studies suggest that these monomeric proteins often possess low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy and guide design of novel monomeric TAL effector-based fusion proteins
Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)
Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through th
Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System
We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications
Efficient genome editing in zebrafish using a CRISPR-Cas system
In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases
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Efficient In Vivo Genome Editing Using RNA-Guided Nucleases
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms
The destruction and survival of dust in the shell around SN 2008S
SN 2008S erupted in early 2008 in the grand design spiral galaxy NGC 6946.
The progenitor was detected by Prieto et al. in Spitzer Space Telescope images
taken over the four years prior to the explosion, but was not detected in deep
optical images, from which they inferred a self-obscured object with a mass of
about 10 Msun. We obtained Spitzer observations of SN 2008S five days after its
discovery, as well as coordinated Gemini and Spitzer optical and infrared
observations six months after its outburst.
We have constructed radiative transfer dust models for the object before and
after the outburst, using the same r^-2 density distribution of pre-existing
amorphous carbon grains for all epochs and taking light-travel time effects
into account for the early post-outburst epoch. We rule out silicate grains as
a significant component of the dust around SN 2008S. The inner radius of the
dust shell moved outwards from its pre-outburst value of 85 AU to a
post-outburst value of 1250 AU, attributable to grain vaporisation by the light
flash from SN 2008S. Although this caused the circumstellar extinction to
decrease from Av = 15 before the outburst to 0.8 after the outburst, we
estimate that less than 2% of the overall circumstellar dust mass was
destroyed.
The total mass-loss rate from the progenitor star is estimated to have been
(0.5-1.0)x10^-4 Msun yr^-1. The derived dust mass-loss rate of 5x10^-7 Msun
yr^-1 implies a total dust injection into the ISM of up to 0.01 Msun over the
suggested duration of the self-obscured phase. We consider the potential
contribution of objects like SN 2008S to the dust enrichment of galaxies.Comment: 9 pages, 7 figures, 3 tables. rv2. To appear in MNRA
Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model
In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications
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