Part:BBa_K1699003
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm
Ribozyme flanked gRNA compatible for dCas9-VP64. gRNA sequence complements 3 different loci in the synthetic promoter pMLPm (2), and gRNA dCas9 complex can promote transcription downstream of syntetic promoter. It has a hammerhead ribozyme on its 5' and an HDV ribozyme on its 3' end. Upon transcription the ribozymes should cleave the mRNA at specific locations to release the mature gRNA (1).
Usage and Biology
guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter dCas9-gRNA complex will promote transcripton of genes downstream of binding site. gRNA scaffold sequence for dCas9 was used (3). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (1).
Characterization
This part has been validated by transfection of human cancer cells HepG2 with dCas9-VP64, this part, and a synthetic promoter with three matching sequences for the gRNA, upstream of GFP. Successfull expression of GFP was observed (Figure 1).
Figure 1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 144
Illegal NgoMIV site found at 173 - 1000COMPATIBLE WITH RFC[1000]
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