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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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Effect of mismatches between crRNA guide and target sequence. (A) Protospacer and crRNA-guide sequences for the B10 target site. Mutations in the 5′ region of the crRNA are shown in lower case; Watson–Crick complementary bases were introduced. (B) Effect of an increasing number of mutations in the 5′ end of the B1, T5 and B10 crRNAs on gfp-mut2 repression mediated by dCas9. Repression by wild-type Cas9 guided by mutant versions of the B10 crRNA is also shown. Fluorescence values are normalized to the fluorescence of a strain expressing gfp-mut2 and dCas9, but no crRNA guide (Ø). Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Effect of Cas9 targeting using a B10 crRNA guide with increasing numbers of mutations at the 5′ end on the transformation of the GFP reporter plasmid, pDB127, or an empty vector control, pZS*24. The mean of three independent enumerations of the total number of colony forming units (CFU) per transformation is shown; error bars indicate one standard deviation. (D) Agarose gel electrophoresis of SacI-digested purified plasmids from cells obtained after transformation of the pDB127-target plasmid into cells expressing wild-type Cas9 and different 5′ mutant versions of the B10 crRNA. Individual plasmids are shown to indicate the electrophoretic mobility of each plasmid.
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gkt520-F2: Effect of mismatches between crRNA guide and target sequence. (A) Protospacer and crRNA-guide sequences for the B10 target site. Mutations in the 5′ region of the crRNA are shown in lower case; Watson–Crick complementary bases were introduced. (B) Effect of an increasing number of mutations in the 5′ end of the B1, T5 and B10 crRNAs on gfp-mut2 repression mediated by dCas9. Repression by wild-type Cas9 guided by mutant versions of the B10 crRNA is also shown. Fluorescence values are normalized to the fluorescence of a strain expressing gfp-mut2 and dCas9, but no crRNA guide (Ø). Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Effect of Cas9 targeting using a B10 crRNA guide with increasing numbers of mutations at the 5′ end on the transformation of the GFP reporter plasmid, pDB127, or an empty vector control, pZS*24. The mean of three independent enumerations of the total number of colony forming units (CFU) per transformation is shown; error bars indicate one standard deviation. (D) Agarose gel electrophoresis of SacI-digested purified plasmids from cells obtained after transformation of the pDB127-target plasmid into cells expressing wild-type Cas9 and different 5′ mutant versions of the B10 crRNA. Individual plasmids are shown to indicate the electrophoretic mobility of each plasmid.

Mentions: Some applications require a precise tuning of gene expression rather than its complete repression. We sought to achieve intermediate repression levels through the introduction of mismatches that will weaken the crRNA/target interactions. We created a series of spacers based on the B1, T5 and B10 constructs that express crRNAs with increasing numbers of mutations in the 5′ end (Figure 2A). Introduction of up to eight mismatches in B1 and T5 did not affect the repression level, and a progressive increase in fluorescence was observed for additional mutations (Figure 2B). A different pattern was observed for the gradual mutagenesis of the B10 crRNA: a gradual decrease in repression was achieved through the introduction of an increasing number of mismatches, with as little as two mismatches providing a lower level of repression than a crRNA fully complementary to the target (Figure 2B). These results demonstrate that the introduction of mismatches in the crRNA guide allows for the modulation of dCas9 repression. However, the number of the mismatches required to achieve this modulation depends on the mode of dCas9 repression, i.e. blocking transcription initiation or elongation.Figure 2.


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)

Effect of mismatches between crRNA guide and target sequence. (A) Protospacer and crRNA-guide sequences for the B10 target site. Mutations in the 5′ region of the crRNA are shown in lower case; Watson–Crick complementary bases were introduced. (B) Effect of an increasing number of mutations in the 5′ end of the B1, T5 and B10 crRNAs on gfp-mut2 repression mediated by dCas9. Repression by wild-type Cas9 guided by mutant versions of the B10 crRNA is also shown. Fluorescence values are normalized to the fluorescence of a strain expressing gfp-mut2 and dCas9, but no crRNA guide (Ø). Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Effect of Cas9 targeting using a B10 crRNA guide with increasing numbers of mutations at the 5′ end on the transformation of the GFP reporter plasmid, pDB127, or an empty vector control, pZS*24. The mean of three independent enumerations of the total number of colony forming units (CFU) per transformation is shown; error bars indicate one standard deviation. (D) Agarose gel electrophoresis of SacI-digested purified plasmids from cells obtained after transformation of the pDB127-target plasmid into cells expressing wild-type Cas9 and different 5′ mutant versions of the B10 crRNA. Individual plasmids are shown to indicate the electrophoretic mobility of each plasmid.
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gkt520-F2: Effect of mismatches between crRNA guide and target sequence. (A) Protospacer and crRNA-guide sequences for the B10 target site. Mutations in the 5′ region of the crRNA are shown in lower case; Watson–Crick complementary bases were introduced. (B) Effect of an increasing number of mutations in the 5′ end of the B1, T5 and B10 crRNAs on gfp-mut2 repression mediated by dCas9. Repression by wild-type Cas9 guided by mutant versions of the B10 crRNA is also shown. Fluorescence values are normalized to the fluorescence of a strain expressing gfp-mut2 and dCas9, but no crRNA guide (Ø). Error bars show one standard deviation from the mean of three relative fluorescence values. (C) Effect of Cas9 targeting using a B10 crRNA guide with increasing numbers of mutations at the 5′ end on the transformation of the GFP reporter plasmid, pDB127, or an empty vector control, pZS*24. The mean of three independent enumerations of the total number of colony forming units (CFU) per transformation is shown; error bars indicate one standard deviation. (D) Agarose gel electrophoresis of SacI-digested purified plasmids from cells obtained after transformation of the pDB127-target plasmid into cells expressing wild-type Cas9 and different 5′ mutant versions of the B10 crRNA. Individual plasmids are shown to indicate the electrophoretic mobility of each plasmid.
Mentions: Some applications require a precise tuning of gene expression rather than its complete repression. We sought to achieve intermediate repression levels through the introduction of mismatches that will weaken the crRNA/target interactions. We created a series of spacers based on the B1, T5 and B10 constructs that express crRNAs with increasing numbers of mutations in the 5′ end (Figure 2A). Introduction of up to eight mismatches in B1 and T5 did not affect the repression level, and a progressive increase in fluorescence was observed for additional mutations (Figure 2B). A different pattern was observed for the gradual mutagenesis of the B10 crRNA: a gradual decrease in repression was achieved through the introduction of an increasing number of mismatches, with as little as two mismatches providing a lower level of repression than a crRNA fully complementary to the target (Figure 2B). These results demonstrate that the introduction of mismatches in the crRNA guide allows for the modulation of dCas9 repression. However, the number of the mismatches required to achieve this modulation depends on the mode of dCas9 repression, i.e. blocking transcription initiation or elongation.Figure 2.

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