Device

Part:BBa_K1150020

Designed by: M. Scheidmann   Group: iGEM13_Freiburg   (2013-09-17)
Revision as of 12:57, 26 September 2013 by Nadine89 (Talk | contribs)

uniCAS Activator (CMV promoter)

We designed a fusion protein consisting of Cas9 and VP16 in order to gain a sequence-specific transactivation of a desired target locus. Therefore we used our double mutated Cas9 (without any cleavage function) and fused it C’terminal with the transactivation domain of VP16.

The whole device includes the CMV promotor, the HA-tag, one N’-terminal NLS, the double-mutated Cas9, a linker of 3 aminoacids, the activation domain of VP16, one C’-terminal NLS and the BGH terminator.

With the help of the HA-tag we can proof the protein expression of our device. Through the usage of two NLS we send our fusion construct into the nucleus. Cotransfecting a RNA plasmid (BBa_K1150034) which includes the tracr and a separately integrated, desired crRNA, the Cas9 specifically binds to the requested DNA sequence. With the help of the transactivation domain of VP16 transcription factors are recruited and the pre-initiation complex can be build. Having targeted this device upstream of a promotor region, it is able to activate the gene of interest.

Fig. 1: Functionality of the fusion protein Cas9-VP19 (BBa_K1150020) for activating gene expression in mammalian cells. The double mutated Cas9 (D10A; H840A) fused to the herpes simplex virus (HSV) derived VP16 activation domain can serve as an crRNA-guided DNA-binding and transactivating protein. If a PAM sequence is present at the 5’ end of crRNA binding site nearly any DNA sequence can be targeted. Abbr.: Pmin: minimal promoter, containing minimal requirements for binding of transcription factors; Cas9: CRISPR associated protein 9; crRNA: CRISPR RNA; tracrRNA: trans-activating RNA; VP16: Virus protein 16, herpes simplex virus (HSV)-derived transcriptional activator protein; PAM: protospacer adjacent motif.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 576
    Illegal BglII site found at 900
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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