Difference between revisions of "Part:BBa K2841513"

Revision as of 13:11, 20 October 2021

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 intermediate LacI repression. 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 LiuP 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).

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 82
Illegal NgoMIV site found at 111
1000
COMPATIBLE WITH RFC[1000]

@@ Line 11: / Line 11: @@
 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 intermediate LacI repression. 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.
-[[File:T--Warwick--SoxSfull.png|400px|right]]<br>
+[[File:T--Warwick--SoxSfull.png|400px|center]]<br>
-Fig.1. Schematic representation of how the added handle would provide a scaffold for the SoxS-MCP CRISPRa system.
+Fig.1. Schematic representation of how the added handle would provide a scaffold for the SoxS-MCP CRISPRa system.<br>
 Activation is far more effective when one of these systems is used: the LiuP 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).