title: Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.
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.
authors.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.
authors.forename: David
authors.initials: D
authors.lastname: Bikard
authors.forename: Wenyan
authors.initials: W
authors.lastname: Jiang
authors.forename: Poulami
authors.initials: P
authors.lastname: Samai
authors.forename: Ann
authors.initials: A
authors.lastname: Hochschild
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Marraffini
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D053263:Gene Regulatory Networks; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D056426:Genetic Loci; D055029:Inverted Repeat Sequences; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012097:Repressor Proteins; D004722:Endoribonucleases