Part:BBa_K2715042
Synthetic guide sequence for S. pyrogenes cas9 - targeting sequence
Usage and Biology
This is a synthetic guide RNA fragment minus the specific 20bp targeting region, designed to work with the Cas9 protein from S. pyrogenes. In our project we aimed to use a nucleolytically inactive Cas9 protein (dCas9) in combination with this guide RNA sequence and a unique 20 bp targeting region to block the transcription machinery from being able to transcribe tcdA in C. difficile, thus reducing its toxicity.
Multiple guide sequences were designed to be used in conjunction with this synthetic guide RNA so that their relative targeting efficiencies could be compared in an assay. The complete list of guides designed can be found below.
Characterisation
The composite parts designed to express this synthetic guide RNA in conjunction with unique targeting regions can be found here BBa_K2715005, BBa_K2715006, BBa_K2715038, BBa_K2715039, BBa_K2715040, BBa_K2715041, and when expressed in conjugation with dCas9, will theoretically reduce toxin expression in C. difficile. This part was characterised using an assay for the reporter GusA in place of the C. difficile toxin A.
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]
References
Huang, H., Chai, C., Li, N., Rowe, P., Minton, N.P., Yang, S., Jiang, W. and Gu, Y., 2016. CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS synthetic biology, 5(12), pp.1355-1361.
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