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

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…

• DR
  Damon Runyon Cancer Research Foundation
• MF
  McKnight Foundation
• LM
  Leona M. and Harry B. Helmsley Charitable Trust
• SS
  Searle Scholars Program
• NI
  National Institutes of Health

  Awards: 1DP2AI104556-01, GM044025