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


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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    • "The only example so far in E. coli uses a fusion to the RNAP ω subunit, with dCas9 targeted approximately 90 base pairs upstream of the transcription start site [33]. Activation is modest (23-fold for a weak promoter, lower effects for stronger promoters) and requires a ΔrpoZ strain but might be improved through fusions with (multiple) alternative factors. "
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    ABSTRACT: Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks which require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and describe efficient strategies for rapidly identifying and correcting causes of failure and fine tuning circuit characteristics.
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    • "For gene activation, it is important to design gRNA in the proximity of the transcription start site at a favorable location in the promoter region to facilitate the easy access of transcriptional factors. For gene repression, gRNA targeting the coding region is more efficient (≥80%) , while there is no repression effect when it binds to antisense strand (Bikard et al., 2013; Choudhary et al., 2015). "
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    ABSTRACT: CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.
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    • "Synthetic DBPs can also function as programmable transcriptional repressors. Localization of dCas9 near transcription start sites (TSSs) can repress active gene expression (Bikard et al. 2013; Qi et al. 2013). However, when fused to repressive domains, such as the KRAB domain (Margolin et al. 1994), the inhibitory effect of ZFs (Beerli et al. 1998), TALEs (Cong et al. 2012), or dCas9 (Gilbert et al. 2013) on transcription is markedly enhanced. "
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    ABSTRACT: Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.
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