Part:BBa_K3081025
pBAD-dCas9-J23119-ctrl-J23119-mRFP
"This composite part is the principal design of the inducible CRISPRi system, with assistance of constantly expressed sgRNA that binds to the complementary coding sequence of mRFP. But the intended sgRNA ctrl can't bind to any sequence of mRFP coding region,so it's used as the control sgRNA. For more detailed information, see BBa_K3081024"
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE 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 5593
Illegal NheI site found at 5616 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1470
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 6176
Illegal AgeI site found at 6288 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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