Part:BBa_K3365000

dCas9-ω

An RNA polymerase ω subunit is fused to the C-terminal of dCas9.

Usage and Biology

The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).

Figure1. Gene circuit

Figure2. Mechanism of CRISPR-mediated transcriptional activation

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1340
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1340
  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1340
  Illegal BamHI site found at 3378
  Illegal BamHI site found at 4212
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1340
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1340
  
• 1000
  COMPATIBLE WITH RFC[1000]