Oct1 Binding Casette 5x UAS

This part contains three times Oct1 recognition sites (BBa_K5237018) and five times an upstream activating sequence (UAS). With these two sequences available we can facilitate binding of Oct1 and Gal4, utilized in our simulated enhancer hijacking using the transactivator fusion protein NLS-Gal4-VP64 (BBa_K5237014). Firefly luciferase will be expressed through Cas staple-induced proximity of the transactivator.

Figure 1: How our part collection can be used to engineer new staples

While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for successful stapling and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome engineering.

Our part collection includes:

1. Sequence overview

2. Usage and Biology

Gal4 is a well-known transcription factor from Saccharomyces cerevisiae that binds specifically to UAS regions on DNA, activating transcription of downstream genes. Its DNA-binding domain has been widely utilized in synthetic biology and gene regulation studies due to its specificity and ability to recruit transcriptional machinery (Kakidani & Ptashne, 1988).
Oct-1 is a key transcription factor that regulates the transcription of the histone H2B gene and various housekeeping genes involved in the cell cycle. As cells transition into mitosis, Oct-1 undergoes hyperphosphorylation, a modification that is reversed when cells exit mitosis. Research shows that a specific phosphorylation site within the homeodomain of Oct-1 is targeted by protein kinase A in vitro. This mitosis-specific phosphorylation is linked to a reduction in Oct-1's DNA-binding ability, both in vivo and in vitro, suggesting that phosphorylation of Oct-1 may contribute to the overall inhibition of transcription that occurs during mitosis ( Segil et al., 1991).
We utilize these two recognition sites for plasmid-to-plasmid stapling with our Cas staples. A fgRNA targeting Oct1 and Tre on the other plasmid, is the key factor in the Cas staple, bringing them together. When the transactivator, Gal4-VP64, binds as well we have transactivation as a readout for functioning staples.

3. Assembly and Part Evolution

The cloning strategy designed for Oct1 allows for the easy assembly of repetitive repeats. It follows the procedure outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI and XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be linearized with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated into the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility. This process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the oligos. For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some limitations regarding sequence depository, the binding cassette is flanked by SalI and XhoI, and the top and bottom oligos with the fitting overhangs are annotated.

4. Results

We were able to show our enhancer plasmid to work great with the Cas staples and the reporter plasmid. For the whole assay, the enhancer plasmid and a reporter plasmid were used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has the Oct1 being targeted by Cas12a. By introducing a fgRNA staple (BBa_K5237000) and a Gal4-VP64 (BBa_K5237021), expression of the luciferase is induced.
Cells were again normalized against ubiquitous renilla expression. Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 2B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene. These results suggest an extension of the linker might lead to better transactivation when hijacking an enhancer/activator.

Figure 2: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by an fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

5. In Silico Characterization Using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PiCasSO system. DaVinci acts as a digital twin to PiCasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA.
We calibrated DaVinci with literature and our own experimental affinity data obtained via EMSA assays and purified proteins. This enabled us to simulate enhancer hijacking in silico, providing valuable input for the design of further experiments. Additionally, we apply the same approach to our part collection.

DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged dna dynamics simulation. Here, we applied the last step to our parts, characterizing structure and dynamics of the dna-binding interaction.

5.1. Enhancer Hijacking is Successfully Studied In Silico

Figure 11: Cas stapled plasmids.

With the Cas staple, we aimed to simulate the principles of enhancer hijacking experiments we conducted in the lab. For these experiments, we modeled the two plasmids also used in the wet lab (BBa_K5237023 and BBa_K5237024). On top of the two plasmids, we modeled the Cas staple by applying a “DNA-DNA tethering force" throughout our simulation, selectively on the regions targeted by the fgRNA. This force was based on simulation data acquired in earlier phases of DaVinci. As there is no suitable model available that also simulates proteins, this proved to be the most effective modeling strategy.

Our simulation showed the expected behavior, holding the target sequences of the Cas staple (Fig. 12). Overall, these exciting results demonstrate that we can successfully model the core principles of enhancer hijacking with a total of 20 thousand simulated nucleotides in silico.

Figure 12: Fusion Cas staples hold stapled nucleotides in close proximity.

5.2. Cas Staple Forces Do Not Distrub DNA Strand Integrity

Figure 13: Cas stapled plasmids.

Next, we aimed to stress test our system to determine the amount of force required to induce DNA double-strand breaks. To achieve this, we used an identical setup to the previous experiment but instead of experimentally determined forces, we used artificial forces of varying strength. It is important to know that our in silico model responds to forces that cause double-strand breaks by scattering the nucleotides across the simulation box. As the specified bonds cannot actually break within the simulation, the phosphate backbone bonds become severely distorted and exhibit erratic behavior in the simulation videos. From our extensive simulations, we concluded that the forces required to damage the DNA are approximately 320 times (Fig. 15) and 680 times greater (Fig. 16) than the forces exerted by the Cas staple, respectively.

This provides important evidence regarding the safety of our staple approach for 3D genome engineering: According to our model, DNA-DNA tethering with our Cas staples is not expected to have a negative effect on the DNA stability.

Figure 14: Applying a force that is 270 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 15: Applying a force that is 320 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 16: Applying a force that is 680 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 17: Applying a force that is more than 1000 times greater
than the predicted force typically exerted by a Cas staple on DNA.

5.3. DaVinci Helps to Design Multi-Staple Arrangements

Figure 18: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.

Finally, we simulated the behavior of multiple staples at once. Therefore, we expanded our previously introduced experimental setup by a second Cas staple.
In a first approach, we targeted an additional region next to the original chosen one. This region is 40 nucleotides away from the first target region on the plasmid displayed in blue connecting it to the opposite site of the orange plasmid, therefore bringing both ends of the orange plasmid together (Fig. 18).
Our simulation predicted that with these staple points, DNA double-strand breaks would occur as clearly visible by the scattered nucleotides (Fig. 19).

Figure 19: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.

Figure 20: Stable multiplexing with 2 Cas staples. On the blue plasmid, the Cas binding sequences are 980 nucleotides apart.

To simulate a setup where we expect no double-strand breaks, we increased the distance between the stapling sites on the blue plasmid (BBa_K5237023) from 40 to 980 nucleotides (Fig. 20).
With this increased distance between stapling sites, we observed a stabilized system. Most interestingly, the non-stapled regions showed maximum distances close to 500 nm, indicating that the two staples led to more compact plasmid structures.

In conclusion, we show that applying multiple staples on the same structures can lead to double-strand breaks if the staples are positioned closely to one another. However, increasing the separation of staples leads to a stable system without DNA damage. Thus, it is definitely possible to multiplex Cas staples, thereby increasing the compactness of DNA structures (Fig. 21). This shows the potential of creating complex regulatory networks by bringing genetic elements into spatial proximity with a multitude of Cas protein staples.

Figure 21: Increasing the distance between Cas staple targets to 980 nucleotides stabilizes multiplexing.

6. References

Kakidani, H., & Ptashne, M. (1988). GAL4 activates gene expression in mammalian cells. Cell, 52, 161-167. https://doi.org/10.1016/0092-8674(88)90504-1.

Segil, N., Roberts, S., & Heintz, N. (1991). Mitotic phosphorylation of the Oct-1 homeodomain and regulation of Oct-1 DNA binding activity. Science, 254(5039), 1814-1816. https://doi.org/10.1126/SCIENCE.1684878.

Sladitschek, H. L., & Neveu, P. A. (2015). MXS-Chaining: a highly efficient cloning platform for imaging and flow cytometry approaches in mammalian systems. PLoS ONE, 10(4), e0124958. https://doi.org/10.1371/journal.pone.0124958.