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

Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA - Nucleic Acids Res. (2013)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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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Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.
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gkt520-F3: Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.

Mentions: We decided to convert dCas9 into a transcription activator. We relied on previous work that demonstrated that a fusion between the lambda cI repressor and the RNAP omega subunit (ω) can activate transcription by stabilizing the binding of RNAP to a promoter bearing an upstream lambda operator (29). Therefore, we made both C- and N-terminal fusions between the ω subunit and dCas9 (Figure 3A). We also expressed different crRNAs to program the binding of both fusion proteins to four different positions in a constitutive synthetic promoter controlling the lacZ gene (Figure 3B) in an E. coli strain lacking the gene encoding for the ω subunit (rpoZ). We used a fusion of the ω subunit to the C-terminus of dCas9 (dCas9-ω lacking a targeting crRNA guide as a control, and we measured β-galactosidase activity as a reporter of gene expression (Figure 3C). The effect of both fusion proteins on lacZ expression depended on the binding site, still causing gene repression when the binding site was too close to the promoter region. With binding sites more distant from the promoter, we observed a modest increase in β-galactosidase activity; in the best case, we observed 2.8-fold activation for the dCas9-ω C-terminal fusion.Figure 3.


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

Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA - Nucleic Acids Res. (2013)

Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753641&req=5

gkt520-F3: Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.
Mentions: We decided to convert dCas9 into a transcription activator. We relied on previous work that demonstrated that a fusion between the lambda cI repressor and the RNAP omega subunit (ω) can activate transcription by stabilizing the binding of RNAP to a promoter bearing an upstream lambda operator (29). Therefore, we made both C- and N-terminal fusions between the ω subunit and dCas9 (Figure 3A). We also expressed different crRNAs to program the binding of both fusion proteins to four different positions in a constitutive synthetic promoter controlling the lacZ gene (Figure 3B) in an E. coli strain lacking the gene encoding for the ω subunit (rpoZ). We used a fusion of the ω subunit to the C-terminus of dCas9 (dCas9-ω lacking a targeting crRNA guide as a control, and we measured β-galactosidase activity as a reporter of gene expression (Figure 3C). The effect of both fusion proteins on lacZ expression depended on the binding site, still causing gene repression when the binding site was too close to the promoter region. With binding sites more distant from the promoter, we observed a modest increase in β-galactosidase activity; in the best case, we observed 2.8-fold activation for the dCas9-ω C-terminal fusion.Figure 3.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

Show MeSH
Related in: MedlinePlus