Part:BBa_K3081044

pBAD-dCas9-ssrA-J23119-NT1-J23119-mRFP

This part is an improvement of BBa_K3081024, which we add a degradation signal peptide ssrA to the dCas9. A more gentle decrease in fluorescence is observed when dCas9 is fused to ssrA tag.

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.

To test the function of fused version of dCas9-ssrA, we co-transformed two plasmids to one E. coli cell, which express mRFP and dCas9-ssrA with sgRNA targeting CDS of mRFP, respectively. As a matter of fact, the new CRISPRi system shows a gentler decrease in fluorescence when dCas9 is fused with ssrA tag, while non-binding dCas9 with or without ssrA has no influence on mRFP expression (Figure 1B). This result confirmed fusion with ssrA, the original function of dCas9 is unaffected.

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 2321
  Illegal NheI site found at 5495
  Illegal NheI site found at 5518
  Illegal NheI site found at 5626
  Illegal NheI site found at 5649
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 4600
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 6209
  Illegal AgeI site found at 6321
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961