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.

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:

DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo
BBa_K5237000	Fusion Guide RNA Entry Vector MbCas12a-SpCas9	Entry vector for simple fgRNA cloning via SapI
BBa_K5237001	Staple Subunit: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
BBa_K5237002	Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
BBa_K5237003	Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004	Staple Subunit: Oct1-DBD	Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005	Staple Subunit: TetR	Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006	Simple Staple: TetR-Oct1	Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007	Staple Subunit: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Staple Subunit: rGCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009	Mini Staple: bGCN4	Assembled staple with minimal size that can be further engineered
Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010	Cathepsin B-cleavable Linker: GFLG	Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011	Cathepsin B Expression Cassette	Expression cassette for the overexpression of cathepsin B
BBa_K5237012	Caged NpuN Intein	A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237013	Caged NpuC Intein	A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237014	Fusion Guide RNA Processing Casette	Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs
BBa_K4643003	IncP Origin of Transfer	Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery
Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237017	FRET-Acceptor: TetR-mScarlet-I	Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237018	Oct1 Binding Casette	DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019	TetR Binding Cassette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020	Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64	Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
BBa_K5237021	NLS-Gal4-VP64	Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022	mCherry Expression Cassette: UAS, minimal Promoter, mCherry	Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker
BBa_K5237023	Oct1 - 5x UAS Binding Casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024	TRE-minimal Promoter- Firefly Luciferase	Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking

1. Sequence overview

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 103
Illegal SpeI site found at 144
Illegal SpeI site found at 175
Illegal SpeI site found at 206
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 144
Illegal SpeI site found at 175
Illegal SpeI site found at 206
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 215
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 103
Illegal SpeI site found at 144
Illegal SpeI site found at 175
Illegal SpeI site found at 206
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 103
Illegal SpeI site found at 144
Illegal SpeI site found at 175
Illegal SpeI site found at 206
1000
COMPATIBLE WITH RFC[1000]

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 and 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 aswell 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 was 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 (see FIGURE results eh panel B). 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 a 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. 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.

Part:BBa_K5237023