Part:BBa_K4367013
dCas9-cTEV
The function of dCas9-cTEV, together with dCas9-nTEV and gRNA, is as a modular DNA sensor that responds by increasing the enzymatic activity of the split TEV if the target DNA is present.
Description
This protein is a fusion of dCas9 and cTEV with a GS-linker (info about TEV: BBa_K4367010). With gRNA, it will be directed towards binding to a specific sequence of DNA, without cutting it like Cas9 would do [1]. With appropriate gRNA, dCas9-cTEV and its counterpart dCas9-nTEV, they would bind close to each other if a specific DNA sequence is present. This would bring nTEV and cTEV much closer to each other on average than if there was no target DNA present, which would cause a greater amount of nTEV and cTEV complementing each other to regain its enzymatic function. This is reasonable as iGEM15_Peking made a similar proof-of-concept, but using split luciferase instead of split TEV bound to dCas9 [2]. Also, iGEM16_Slovenia made a proof-of-concept that TEVp could be split and undergo complementaion [3]. In conclusion, the presence of a specific DNA sequence would induce a higher enzymatic activity of the split TEV in the dCas9-splitTEV system. This is illustrated in figure 1.
Figure 1. The dCas9-splitTEV system binding to DNA and inducing complementation of split TEV.
Usage
The dCas9-splitTEV system is used as a DNA sensor which will activate a reporter protein that responds to the enzymatic activity of TEV. One reporter is the FRET Protein (FP: BBa_K4367011), which is a pair of fluorescent proteins bound together by a cleavable linker. If the linker is cleaved by TEV, the FRET phenomnea will disappear, which would be measurable with appropriate lab equipment. The other reporter protien is Inhibited Beta-galactosidase (iGal: BBa_K4367009), which will undergo Alpha-complementation when cleaved by TEV and produce a blue pigment visible by eye.
The dCas9-splitTEV system alongside iGal is illustrated in figure 2.
Figure 2. The Cell-free Modular DNA Detection Device. dCas9-splitTEV binds to DNA and activates iGal to produce a visible readout.
Future design considerations
The GS-linker in the part is 20 amino acids long, although that may or may not be optimal depending on the distance between where the pair of dCas9 binds. A longer distance would reasonably result in a longer optimal linker, but that relationship would have to be established experimentally. Our software, PLOP, uses a model to predict the likelyhood of the halves of the split TEV being in close proximity, which could give a clue to what types of gRNA and length of linkers would be reasonable to test. We intended to test multiple linkers and gRNA to test our model and find an optimal combination of these, but that turned out to require more time than our timeframe allowed - The potential of this system has not yet been explored.
It was elaborated in the part TEVp (BBa_K4367010) that there exists multiple possible proteases that have specific recognition sites and can be split like TEVp [4,5]. The following proteins were simulated in AlphaFold with promising results: dCas9-nPPV, dCas9-nSbMV, dCas9-nSuMMV, dCas9-nHRV_3C, and dCas9-cHRV_3C. The simulations are shown in figure 3 and figure 4. These simulations displayed that the folding of dCas9 was correct, and that the split protease was essentially free floating outside of dCas9 but still anchored to it with a linker.
Figure 3. Simulation in AlphaFold on the fusion between dCas9 and the following proteases: nPPVp (yellow), nSbMV (red) and nSuMMV (blue).
Figure 4. A simulation in AlphaFold where nHRV_3C (red) and cHRV_3C (cyan) are bound to dCas9.
A simulation on nTEV-dCas9 and cTEV-dCas9 (opposite order as in the parts) showed worrying results that the split TEV may distrub the folding of dCas9 by binding to the insides of dCas9. It is unclear how representative this result is for dCas9-nTEV and dCas9-cTEV (observe the order!), but it would be a good idea to simulate these in AlphaFold if one were to use this part. If the results are still worrying, it would reasonable to examine the alternative proteases.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4358
Illegal BamHI site found at 4687 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 14
Illegal BsaI.rc site found at 4694
Illegal SapI.rc site found at 4594
References
[1] Chengkun Wang, Yuanhao Qu, Jason K. W. Cheng, Nicholas W. Hughes, Qianhe Zhang, Mengdi Wang & Le Cong (2022).dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Available at: https://www.nature.com/articles/s41556-021-00836-1
[2] iGEM15_Peking (2015). PC Reporter. Available at: https://2015.igem.org/Team:Peking/Design/PC_Reporter
[3] iGEM16_Slovenia (2016). Split proteases. Available at: https://2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases
[4] Tina Fink, Jan Lonzarić, Arne Praznik, TjaÅ¡a Plaper, Estera Merljak, Katja Leben, Nina Jerala, Tina Lebar, Žiga StrmÅ¡ek, Fabio Lapenta, Mojca BenÄina & Roman Jerala (2019). Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Available at: https://www.nature.com/articles/s41589-018-0181-6 iGEM16_Slovenia has been a source inspiration in the design of the Cell Free system and they were the ones that came up with the proof-of-principle behind the paper ‘Design of fast proteolysis-based signaling and logic circuits in mammalian cells’.
[5] Shengchen Wang, Faying Zhang, Meng Mei, Ting Wang, Yueli Yun, Shihui Yang, Guimin Zhang & Li Yi (2021). A split protease-E. coli ClpXP system quantifies protein–protein interactions in Escherichia coli cells. Available at: https://www.nature.com/articles/s42003-021-02374-w
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