Part:BBa_K1723012:Design
c3_3 gRNA expressing sequence
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 145
Illegal NgoMIV site found at 174 - 1000COMPATIBLE 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_3 gRNA - dCas9_VP64 complex works as an activator of the RNA Polymerase II CYC_3 (BBa_K1723025) 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)