RNA

Part:BBa_K1818000:Design

Designed by: Connor McBrine   Group: iGEM15_Tufts   (2015-09-18)

T7-BbsI-gRNA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The T7 promoter starts transcription at it second to last base pair. This means all transcripts will start with GG. The genomic target should therefore also start with GG (not to be confused with the distal NGG protospacer adjacent motif, which is also essential). A protocol for oligo design and insertion can be found here: http://flycrispr.molbio.wisc.edu/protocols/gRNA


Source

The T7 promoter is from T7 bacteriophage, whereas the guide RNA is a chimeric sequence developed in the Doudna lab at UC Berkeley. It is meant to mimic the natural crRNA:tracrRNA complex. The overall design is from the labs of Melissa Harrison, Kate O'Connor-Giles, Jill Wildonger who created the pU6-BbsI-chiRNA plasmid for generating guide RNAs using a Drosophila promoter.

References

Jinek, M, Chylinski, K, Fonfara, I, Hauer, M, Doudna, J, Charpentier, E. (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821.

flyCRISPR. (2013). http://flycrispr.molbio.wisc.edu/protocols