Part:BBa_K4212005
gRNA_SDP
Designed for CRISPR system introducing double stranded cut in origin of replication of BBa_K4212031 part (Golden Gate level 2 B. subtilis expression vector). Useful for production of self-digesting plasmid system functional in B. subtilis.
Usage and Biology
In order for Cas9 to introduce a double stranded break in the DNA and hence unlock the nuclease activity of D15, a gRNA sequence was designed to target the origin of replication of the plasmid hosting the self digesting circuitry. The rationale behind this approach lies in the mode of action of D15. Indeed, this exonuclease hydrolyses phosphodiester bonds of nucleotide chains starting from their 3’ end. In order to increase the efficacy of our system, we hypothesized that a cut in the ori impair the functionality of the replicative region, hence limiting plasmid retention and duplication post-sporulation. As a matter of fact, even limited digestion of this regulatory region could still lead to the inability of replication of our system. This would still allow to reach appropriate protein expression levels while avoiding failure in digesting activity towards plasmid circuitry.
References
[1] Kiani, S., Chavez, A., Tuttle, M., Hall, R.N., Chari, R., Ter-Ovanesyan, D., Qian, J., Pruitt, B.W., Beal, J., Vora, S., Buchthal, J., Kowal, E.J.K., Ebrahimkhani, M.R., Collins, J.J., Weiss, R. & Church, G. (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nature Methods. 12 (11), 1051–1054. doi:10.1038/nmeth.3580.
[2] Wilson, L.O.W., O’Brien, A.R. & Bauer, D.C. (2018) The Current State and Future of CRISPR-Cas9 gRNA Design Tools. Frontiers in Pharmacology. 9. https://www.frontiersin.org/articles/10.3389/fphar.2018.00749.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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