Difference between revisions of "Part:BBa K1699003"

Revision as of 10:32, 13 September 2015

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 Pol 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 144
Illegal NgoMIV site found at 173
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1699003 short</partinfo>
-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).
+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 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).
+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 Pol III promoters, like U6 promoter, which is generally used to synthesize gRNAs.
 ===Characterization===