Part:BBa_K5237023

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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.

1 Sequence overview
2 Usage and Biology
3 Assembly and part evolution
4 Results
5 References

The PICasSO Toolbox

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

Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation, cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our Simple staples that 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 the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of 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 for functional readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking 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.

Our part collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000	fgRNA Entry vector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Staple subunit: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple
BBa_K5237002	Staple subunit: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
BBa_K5237003	Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA 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. Can be used to create functionalized staples units
BBa_K5237013	Caged NpuC Intein	A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as 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 to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. 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. 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. It 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 Promotor, mCherry	Readout system for enhancer binding. It 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. It 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.

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