Part:BBa_K1699003
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm
Ribozyme flanked gRNA compatible for dCas9-VP64. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of synthetic promoter. It has a hammerhead ribozyme on its 5' and an HDV ribozyme on its 3' end. Upon transcription the ribozymes cleave the RNA at specific locations to release the mature gRNA (2, 3).
Usage and Biology
Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 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-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for SpdCas9-VP64 was used (4). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (2, 3). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation, while eliminating the need for use of constitutuve RNA Polymerase III promoters, like U6 promoter, which is generally used to synthesize gRNAs.
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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