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Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA, Department of Microbiology and Immunobiology, Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115, USA, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nucleic Acids Research (Impact Factor: 9.11). 06/2013; 41(15). DOI: 10.1093/nar/gkt520
Source: PubMed

ABSTRACT The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

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Available from: David Bikard, Sep 05, 2014
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    • "By supplying donor templates comprising exogenous sequence flanked by homology-containing stretches (commonly referred to as homology arms), the HDR pathway can be appropriated to make precise modifications including defined deletions, sequence substitutions, or insertions. Beyond the genome engineering applications of the CRISPR-Cas9 system, nuclease-dead Cas9 is being used as a sequence-specific repressor or activator of gene expression, and developed as a tool for probing genome structure and function without causing mutations (Bikard et al., 2013; Chen et al., 2013; Cheng et al., 2013; Fujita and Fujii, 2013; Gilbert et al., 2013; Maeder et al., 2013; Perez-Pinera et al., 2013; Qi et al., 2013; Anton et al., 2014; Kearns et al., 2014). "
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    ABSTRACT: The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 01/2015; 111:31.2.1-31.2.20. DOI:10.1002/0471142727.mb3102s111
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    • "Recent work demonstrated that a catalytically inactive Cas9 protein (dCas9) could be combined with guide-RNAs and used as a synthetic DNA-targeting module, because the binding of dCas9 to selected targets interfered with gene transcription (Bikard et al., 2013; Qi et al., 2013). Precisely targeted mutations of the two Cas9 endonuclease domains, RuvC D10A and HNH domain H841A, converted the prokaryotic CRISPR/Cas system from a sitespecific genome-editing tool to a specifically targeted genomeregulation tool. "
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    ABSTRACT: Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.
    Plant Biotechnology Journal 11/2014; 13(4). DOI:10.1111/pbi.12284 · 5.68 Impact Factor
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    • "Besides dsDNA cleavage, the CRISPR-Cas system has been recently modified for nicking and repression of gene activity [10]. It is reported as an effective tool for activation of gene expression also [11]. Due to these novel features, this system is going to play very important role in genome engineering programs. "
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    ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system facilitates targeted genome editing in organisms. Despite high demand of this system, finding a reliable tool for the determination of specific target sites in large genomic data remained challenging. Here, we report SSFinder, a python script to perform high throughput detection of specific target sites in large nucleotide datasets. The SSFinder is a user-friendly tool, compatible with Windows, Mac OS, and Linux operating systems, and freely available online.
    BioMed Research International 06/2014; DOI:10.1155/2014/742482 · 2.71 Impact Factor
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