The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. 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 with our interkingdom conjugation system.

(iii) As the final component 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 readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

The following table gives a complete 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 parts 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 Entryvector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Half-Staple: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9
BBa_K5237002	Half-Staple: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
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 in close proximity
BBa_K5237004	Half-Staple: 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	Half-Staple: 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	Half-Staple: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Half-Staple: 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	Cathepsin B which can be selectively express to cut the cleavable linker
BBa_K5237012	Caged NpuN Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237013	Caged NpuC Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
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	Donor part for the FRET assay binding 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, can be used for different 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 - UAS binding casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.
BBa_K5237024	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

2. Usage and Biology

The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell. TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth et al. 2000; Kisker et al. 1995).
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in synthetic biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its well-characterized behavior, ensuring reliable DNA interactions.

3. Assembly and part evolution

TetR was C-terminally fused to create a tetR-mScarlet-I-His₆.

4. Results

The fusion protein was expressed from a T7 based expression plasmid and subsequently purified using metal affinity chromatography with Ni-NTA beads.(Figure 1, left) DNA binding affinity in two different buffer systems was estimated with an electrophoretic mobility shift assay (EMSA) (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl).

Figure 2: Expression and DNA binding analysis of tetR-mScarlet-I-His₆ fusion protein.
Left image: SDS-PAGE analysis of protein expression. Lane 1: raw lysate of E. coli expression culture after steril-filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na₂HPO₄, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na₂HPO₄, 150 mM NaCl, 20 mM Imidazol)); Lane 4: Elution of purified protein. The expected band size of the protein is 50 737.60 Da, highlighted with a red box on the gel.
Right image: Qualitative electrophoretic mobility shift assay of tetR in two different buffer systems. 1 µM protein and 0.5 µM DNA containing three tetR binding sites were equilibrated in different buffer sytstems (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl). Bands were visualized by SYBR-safe staining after gel electrophoresis

5. References

Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), Genetic Engineering: Principles and Methods (pp. 255-277). Springer US. 10.1007/978-0-306-48573-2_13

Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor and Tetracycline-Mg2|ihsbop|+Reveals Mechanism of Antibiotic Resistance. Journal of Molecular Biology, 247(2), 260-280. 10.1006/jmbi.1994.0138

Saenger, W., Hinrichs, W., Orth, P., Schnappinger, D., & Hillen, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural & Molecular Biology, 7(3), 215-219. 10.1038/73324

References

Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), Genetic Engineering: Principles and Methods (pp. 255-277). Springer US. 10.1007/978-0-306-48573-2_13

Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor and Tetracycline-Mg2|ihsbop|+Reveals Mechanism of Antibiotic Resistance. Journal of Molecular Biology, 247(2), 260-280. 10.1006/jmbi.1994.0138

Saenger, W., Hinrichs, W., Orth, P., Schnappinger, D., & Hillen, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural & Molecular Biology, 7(3), 215-219. 10.1038/73324