Part:BBa_K2841513
Self-Cleaving gRNA Kit
A fusion of BBa_K2841512 and BBa_K2841511. A self-terminating self-cleaving gRNA cis acting factor (a guide RNA without the targeting region.)
Simply put the complementary sequence of the DNA that you would like the gRNA to target just before this biobrick part, and express BBa_K2841510. The dCAS9 will localise to your target. You do not need a terminator - this part will self terminate!
Warwick 2021 Contribution
CRISPRa The handle exclusively works as a CRISPRi system. iGEM 2018 used it as a double not-gate to act as CRISPRa, needed intermediate LacI repression.
Speed By changing the handle with either of these two papers, we can make it into a CRISPRa. As per the data of the iGEM 2018 team, and per the data of the papers, we can change the induction time from days (with the not-gate system) to hours
Stability - the error bars are further away, and smaller
Flexibility - multiple gRNA spacer targets rather than just one protein, much more open in what can be enhanced.
References
[1] - Liu, Y., Wan, X. & Wang, B. Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria. Nat Commun 10, 3693 (2019). https://doi.org/10.1038/s41467-019-11479-0 [2] - Dong, C., Fontana, J., Patel, A. et al. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria. Nat Commun 9, 2489 (2018). https://doi.org/10.1038/s41467-018-04901-6 [3] - Roberto Galizi, John N. Duncan, William Rostain, Charlotte M Quinn, Marko Storch, Manish Kushwaha, and Alfonso Jaramillo.The CRISPR Journal.Oct 2020.398-408.http://doi.org/10.1089/crispr.2020.0029
Sequence and Features
- 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 82
Illegal NgoMIV site found at 111 - 1000COMPATIBLE WITH RFC[1000]
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