Coding

Part:BBa_K1699005

Designed by: Emil Ruvinov   Group: iGEM15_BGU_Israel   (2015-08-09)

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 promoter

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under the control of U6 (RNA polymerase III) promoter. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of a synthetic promoter.


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 Sp dCas9-VP64 was used (2).

Characterization

This part was designed as a control construct for two cancer-specific promoter-based design of CRISPR-mediated transcriptional activation. In the design, gRNA is ribozyme-flanked (to enable synthesis under RNA polymerase II promoter) and is under control of human survivin promoter.

U6-gMLP: AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human U6 promoter (Fig. 1).

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Fig. 1. Plasmid map of AAV vector carrying gMLP under the control of human U6 promoter.

References

1. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. http://www.ncbi.nlm.nih.gov/pubmed/23977949

2. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25. http://www.ncbi.nlm.nih.gov/pubmed/23892895


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 AgeI site found at 250
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None