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.
    Journal of Molecular Biology 10/2015; DOI:10.1016/j.jmb.2015.10.004 · 4.33 Impact Factor
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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.
    Current issues in molecular biology 10/2015; 20:1-12. · 5.75 Impact Factor
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    • "Modified versions of the Cas9 protein have been engineered by mutating important catalytical residues, generating Cas9 proteins that cut only one DNA strand (nickase Cas9) or that are completely inactive (Jinek et al., 2012). Catalytically inactivated Cas9 proteins (dead Cas9, dCas9) can be used to control gene expression, either by physically interfering with the transcription process (Gilbert et al., 2013; Qi et al., 2013) or as fusion proteins with factors that mediate transcriptional repression or activation (Bikard et al., 2013; Cheng et al., 2013; Gilbert et al., 2013; Hu et al., 2014; Maeder et al., 2013a; Mali et al., 2013b; Perez-Pinera et al., 2013). These systems have recently been utilized to mediate target gene activation and repression in mouse and human cells to promote differentiation of human cells (Chavez et al., 2015; Kearns et al., 2014) and transdifferentiation and reprogramming of mouse cells (Chakraborty et al., 2014; Gao et al., 2014). "
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    ABSTRACT: CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
    Stem Cell Reports 09/2015; 5(3):448-459. DOI:10.1016/j.stemcr.2015.08.001 · 5.37 Impact Factor
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