Difference between revisions of "Part:BBa K1723000:Design"
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<partinfo>BBa_K1723000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1723000 SequenceAndFeatures</partinfo>
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===dCas9-ω===
 
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Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease that targets and cleaves any DNA sequence complementary to its guide RNA (gRNA). Our project will be based upon a derivative of this technology : catalytically “dead” Cas9 (dCas9) that lack the ability to cleave DNA. When fused to a RNA polymerase (RNAP) recruiting element (e.g. the omega subunit of RNAP in E. Coli or VP64 in eukaryotes), chimeric dCas9 can act as a  programmable transcription activator. In addition, activating dCas9 may also act as a DNA transcription inhibitor: depending on its gRNA-determined binding site, it has been shown in yeasts to sterically hinder RNAP recruitment to promoter sequences. [1]
 
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[1] Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437.
 
  
  
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===Source===
 
===Source===
  
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A plasmid containing dCas9_&#969; (pdCas9) was given to our iGEM team by David Bikard.
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The omega subunit was extracted from the plasmid pWJ66 given to the iGEM15_EPFL team by David Bikard.
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The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249)
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===References===
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Latest revision as of 21:25, 17 September 2015

dCas9-ω


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1099
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3378
    Illegal BamHI site found at 4212
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

dCas9 is a Cas9 double mutant, with mutations at amino acid positions D10A and H840A. These mutations inactivate Cas9 nuclease and nickase activities. Cas9-ω contains an EcoRI restriction site which was removed via site-directed mutagenesis. Fusion of the omega subunit (rpoZ) to dCas9 was achieved by Gibson assembly.

Source

The omega subunit was extracted from the plasmid pWJ66 given to the iGEM15_EPFL team by David Bikard. The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249)