Team:EPF Lausanne/Description
Bikard et al. used dCas9-ω in order to regulate gene expression [1]. Their dCas9 system works with tracRNAs, while our system works with simpler gRNAs. We also added to the iGEM Parts Registry Bikard's promoter, BBa_K1723001, which is an improved version of the J23117 (BBa_J23117) promoter. In addition, to test new gRNA sequences we created a fully synthetic biobrick, BBa_K1723005, which is a mutated version of Bikard and al.'s promoter.
dCas9-ω
This part can be considered as an improvement of the biobrick dCas9-ω Activator (BBa_K1218014) . Our protein acts using sgRNAs (single guide RNA, such as BBa_K1723002) as guide RNAs [2], instead of tracrRNA/CRISPR array system. SgRNAs are more modular as they can be produced separately when needed in CRISPRi context. Also, the production of the complex is faster as less processing steps are needed. Finally, the sequences are shorter and facilitate the scalability.
PAM rich URS J23117 promoter
PAM rich URS J23117 is an improvement of the J23117 (BBa_J23117) weak promoter. This promoter is the promoter J23117 flanked with a PAM (PAM = NGG sequence) rich Upstream Regulatory Sequence (URS) to enable the use of protein dCas9-ω (BBa_K1723000) as a gene transcription regulator.
PAM rich URS J23117Alt promoter
PAM rich URS J23117Alt is a new fully synthetic promoter obtained by mutation of PAM rich URS J23117 promoter (BBa_K1723001). It acts the exactly same way as the PAM rich URS J23117 promoter but is targeted by others specific sgRNAS.
References
[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.
