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 in its original form exclusively works as a CRISPRi system. iGEM 2018 used it as a double not-gate to act as CRISPRa, needing immediate LacI repression [3]. By changing the handle with either from papers [1] or [2] and expressing the respective transcriptional activator (LiuP or SoxS-MCP, respectively - example for the latter in Fig.1), it can be turned into a CRISPRa component. As per the data of the iGEM 2018 team, and per the data of the papers, the induction time can be changed from days (with the not-gate system) to hours.
Fig.1. Schematic representation of how the added handle would provide a scaffold for the SoxS-MCP CRISPRa system.
Activation is far more effective when one of these systems is used: the PspF system presents a 78-fold increase in fluorescence when a fluorescent reporter is activated with the system as opposed to a mismatching gRNA (Fig.2).
Fig.2. Graph indicating the 78-fold increase in expression of a reporter when the gRNA is modified to possess a LiuP binding handle and PspF and dCas9 are all expressed in the examined cells [1].
Such a system would also be highly reliable as there appears to be little variability in the data according to the cited studies. Moreover, the great amount of modularity different activator protein binding handles (as seen in Fig.3) offer can be used in combining different systems in the same cells when cross-activation is not desirable. In addition this allows the use of different activators for different sequences, allowing the best one for each individual target to be picked without much effort.
Fig.3. NUPACK simulations of the secondary structures of the different cgRNAs. Note the differently-shaped boxB (for PspF[1]) and MS2 (for SoxS-MCP[2]) binding handles - off-target binding of these proteins is highly unlikely, allowing specificity of each system to their respective targets.
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. Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics, 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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