Part:BBa_K3081024

pBAD-dCas9-J23119-NT1-J23119-mRFP

This composite part is the principal design of the inducible CRISPRi system, with assistance of constantly expressed sgRNA (NT1) that is complementary to the corresponding sequence of mRFP coding region. Fluorescence is decreased as the concentration of arabinose increases.

To investigate CRISPR-dCas9 binding specificity and affinity with DNA, we constructed arabinose-induced expression of dCas9 targeted to mRFP coding region, with assistance of constantly-expressed single guide RNA that is complementary to the corresponding sequence. Since normal mRNA elongation is interrupted by occurrence of dCas9, fluorescence is greatly decreased as the arabinose concentration increases, comparing to a single guide RNA that has no binding specificity to DNA. This proves the basic concept that a dCas9 protein is able to bind to DNA with sequence specificity and interferes with the physiological process.

Figure 1. Comparison between dCas9 and dCas9-ssrA system by CRISPRi effect on mRFP fluorescence.

Reference:

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 5459
  Illegal NheI site found at 5482
  Illegal NheI site found at 5590
  Illegal NheI site found at 5613
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1470
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 6173
  Illegal AgeI site found at 6285
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961