RNA

Part:BBa_K1723010:Design

Designed by: Cyril Pulver   Group: iGEM15_EPF_Lausanne   (2015-09-16)

c3_1 gRNA expressing sequence


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 145
    Illegal NgoMIV site found at 174
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The Hammerhead ribozyme - gRNA - HDV rizobyme sequence design allows expressing gRNAs using a RNA polymerase II promoter which can be regulated contrary to RNA polymerase III promoters [1]. This is much needed when gRNAs need to be produced on demand, like in a logic system.

This design also makes it possible to express several gRNAs in the same RNA transcript, as they will free themselves from the transcript thanks to both self-cleaving ribozymes.

For example: a first gRNA is produced, forms a complex with dCas9_VP64 (BBa_K1723021) which binds to a RNA Polymerase II promoter controlling a second gRNA expressing sequence, thus producing the second gRNA.

The c3_1 gRNA - dCas9_VP64 complex works as an activator of the RNA Polymerase II CYC_01 (BBa_K1723023) promoter. A similar mechanism has been proven to work with gRNA c3_0 (produced by BBa_K1723009) and promoter CYC_0 (BBa_K1723022) [2].

Note that the 20 bp SDS sequence can be changed to produce any other gRNA at will.


Source

Synthesized as a G-Block.

References

[1] Gao, Y., and Zhao, Y. (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56 , 343–349

[2] Farzadfard, F., Perli, S.D., Lu, T.K., 2013. Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas, ACS Synthetic Biology (ACS publications), http://pubs.acs.org/doi/pdf/10.1021/sb400081r (16.09.2015)