Tools Design

The project aims to study how bacterial DNA organization can influence gene expression. In order to answer this question, we decided to create a new tool based on CRISPR/Cas9 technology to bring together two DNA regions separated in space. This new tool is expected to assess the effect of DNA structure on gene expression. We designed a bring DNA closer tool (BDC tool) and a visualization tool.

In this part, we expose our tool design, our experimental strategy is available here.

Molecular tools

In order to design our two tools, we need specific molecular tools with specific functions: DNA recognition, DNA binding function, dimerization and a fluorescence system.

DNA recognition and fixation

The two tools have to recognize a specific target sequences on DNA and then bind to it. Several options were possible, such as a protein-DNA or a RNA-DNA recognition. First of all, we thought to use engineered nuclease proteins, such as zing-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or a RNA-guided engineered nuclease (RGEN) for their ability to target a specific sequence thanks to a specific proteic or nucleic sequence (for TALEN for instance, we speak about TALE code). We chose a based CRISPR/Cas9 recognition system but for the Cas9 protein, we chose an endonuclease deficient Cas9 (dCas9). For more information about CRISPR/Cas9 technology, click here. The CRISPR/Cas9 system is a quite new tool for synthetic biology which has many applications, such as targeting specific DNA sequences. Nevertheless, our system allows a new application of CRISPR/Cas9 technology. We chose dCas9 for three reasons :

• simplicity : to target DNA sequence, we only have to design a specific sgRNA, which takes about a week
• adaptability : the tool would be adaptable to target any sequences in the genome through the sgRNA sequence design
• specificity : the use of orthologous dCas9s allows a specific one by one sgRNA/dCas9 recognition by a specific PAM sequence for each pair

Dimerization system

Our project aims to recognize specific sequences on the genome and then induce a spatial proximity. So once we chose a system allowing a DNA targetting, we had to chose a system which could dimerize the dCas9/sgRNA, in order to get these two dCas9/sgRNA closer. We chose the induced dimerization FRB/FKBP12 system. In fact, this system allows a dimerization induced by rapamycin. To avoid any problem with rapamycin and E. coli, we decided to used an analog of rapamycin called rapalog (gratefully offered by Takara Clonethec). We then used a FRB mutated protein sequence (FRB*) to be recognized by rapalog.

Fluorescence system

For the visualization tool, we needed a fluorescence system to asses a spatial proximity due to our system. We chose the tripartite split-GFP, which is divided in three different parts: two twenty amino acids parts corresponding to the 10^th and 11^th beta-sheets and a third part corresponding to the other beta-sheets of the GFP. This solution avoids poor folding and/or self-assembly background fluorescence. With this system, only two sgRNAs/dCas9s fused to their specific GFP tags are necessary to obtain an accurate fluorescence.

Bring DNA Closer tool construction

We have designed a tool based on CRISPR/Cas9 property to target a specific DNA sequence. We imagined a system using dCas9 that dimerizes under an induction signal to bring two DNA sequence closer. A dCas9 is a protein which recognize precisely a DNA sequence with dead nuclease activity. We choosed it for the high adaptability of this system, as it target DNA through a sgRNA it is easy to customize the target sequence. But as we need to target two different sequences we also need to work with dCas9s which will not interfere with each other. So we choosed two orthologous dCas9s which come from two different organisms T. denticola (TD) and S. pyogenes (SP). As they come from different organisms they recognize different sgRNAs and do not interfere as we wanted. We order from Addgene the plasmid coding for each one of these dCas9s and their sgRNAs.

To dimerize this two dCas9s we have chosen an inducible system using FRB and FKBP12 proteins. Originally found in mammals this two proteins form an heterodimer when rapamycin is added, it is particularly used in protein interaction studies (Cui et al., 2014). However rapamycin is toxic for bacteria. But studies have shown that a mutated FRB (FRB*) stills allow dimerization with an analog of rapamycin non toxic called rapalog. The mutations implied are: T2098L, K2095P, W2101F.(Bayle et al., 2006; Liberles, Diver, Austin, & Schreiber, 1997).

A biobrick coding FRB with mutation T2098L was already in the parts registry (iGEM Part_ J18926) but it was not available. Moreover it contains only one mutation on the 3 described in the literature. So we decided to work with the fully mutant FRB. Rapalog and plasmid with mutant FRB and FKBP12 were offered to us by Takara Clontech. But like we mentioned previously this system is used in mammal cells, so we decide to optimize the sequences for an expression in E.coli with the Jcat plateforme. So we finally order gBlocks coding for the FRB* and FKBP12 optimized sequences and a linker in prevision to the fusion with their respective dCas9s.

Using these two systems (dCas9 recognition and FRB/FKBP12 dimerization) we design our new tool based on the two following BioBricks:

These two biobricks will be assembled in pSB1C3 plasmid giving us our BDC tool which will function as bellow:

Visualization tool construction

Every system need an efficient control, as a result, a new part of the project has been setting up and the team has designed the visualization tool.

To build this new tool, two other orthologous nuclease function deficient Cas9s (dCas9s): N.meningitidis and S.thermophilus, will be fused to fluorescent proteins. We also decide to use dCas9 system for this tool in order to have detection of a accurate and unique sequence in the genome. It will be fulfilling with a new and unreleased in the iGEM competition tripartite slip-GFP.

The tripartit split-GFP is composed of two twenty amino-acids long GFP tags (GFP10 and GFP11) and a third complementary subsection (GFP1-9). The tags will be fused to the two dCas9 previously quoted. A functional GFP will be achieved when the tools would be close enought to allow the three slip-GFP parts reunion and the fluorescence emission. This fluorescence system avoids poor folding and/or self-assembly background fluorescence. With this system, only two sgRNAs associate with their dCas9s fused to their specific GFP tags will be necessary instead of nearly 30 with mundane GFP due to background fluorescence.

The team has designed three biobricks to achieve this part of the project:

• BioBrick n°3 : N.meningitidis fused to GFP-10 expressed by a constitutive promoter, a RBS and a double terminator
• BioBrick n°4 : S.thermophilus fused to GFP-11 expressed by a constitutive promoter, a RBS and a double terminator
• BioBrick n°5 : The third part of the GFP 1-9 expressed by a constitutive promoter, a RBS and a double terminator

The biobricks were inserted into pSB1C3 using the iGEM process : restriction sites EcoRI and PstI.

Then, the team has considered to establish a composite biobrick composed of the three biobricks in the same pSB1C3 plasmid. This plasmid would have been build using the iGEM restriction site technique.

The dCas9 technique allows the target of specific sequences. In fact, this technique its adaptable to any DNA sequences on the genome through the single guide RNA (sgRNA). Those sgRNAs are associated with their cognate ortholog dCas9s mostly thanks to their palindromic associate motif (PAM).

The team has chosen to express the sgRNAs on another plasmid pZA11 which is compatible with pSB1C3.

References

Bayle, J. H., Grimley, J. S., Stankunas, K., Gestwicki, J. E., Wandless, T. J., & Crabtree, G. R. (2006). Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chemistry and Biology, 13(1), 99–107. http://doi.org/10.1016/j.chembiol.2005.10.017

Chen, Baohui et al. 2013. “Resource Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System.” CELL 155(7): 1479–91. http://dx.doi.org/10.1016/j.cell.2013.12.001.

Church, George M. 2014. “HHS Public AccessOrthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing.” Nat. Methods 10(11): 1116–21.

Cui, B., Wang, Y., Song, Y., Wang, T., Li, C., Wei, Y., … Shen, X. (2014). Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria. mBio, 5(3), 1–10.http://doi.org/10.1128/mBio.01050-14

Liberles, S. D., Diver, S. T., Austin, D. J., & Schreiber, S. L. (1997). Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proceedings of the National Academy of Sciences of the United States of America, 94(15), 7825–7830.http://doi.org/10.1073/pnas.94.15.7825

Ma, Hanhui et al. 2015. “Multicolor CRISPR Labeling of Chromosomal Loci in Human Cells.” Proceedings of the National Academy of Sciences of the United States of America.

Nguyen, Hau B et al. 2013. “A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association.” Scientific Reports 10: 1–9.