Part:BBa_K5237012
Caged NpuN Intein
The Caged NpuN Intein is derived from the naturally split intein DnaE of the cyanobacterium Nostoc punctiforme, designed to facilitate controlled protein trans-splicing. By caging the N- and C-terminal intein fragments (NpuN and NpuC), splicing is inhibited until removal of the cages, allowing precise regulation of protein linkage. The caged NpuN intein fragment was codon optimized for expression in human cells, with modifications to the NpuC sequence for enhanced functionality. The system enables the conditional assembly of proteins, such as the oligomerization of dead Cas9, via cathepsin B-mediated cleavage, providing a versatile tool for synthetic biology applications.
Contents
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 parts 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 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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2. Usage and Biology
Inteins are protein sequences that splice themselves out of a polypeptide chain through an autocatalytic cleavage reaction. This process ligates the flanking polypeptides, termed exteins (Mills, Johnson & Perler, 2014; Wang et al., 2022). Some inteins are naturally split in two parts – termed N- and C-terminal intein fragments. Trans-splicing of two split intein fragments can covalently link two different proteins (Ventura & Mootz, 2019).
The naturally split intein DnaE from the cyanobacterium Nostoc punctiforme (Npu) was previously utilized to link different protein fragments in prokaryotic and eukaryotic systems (Gramespacher et al., 2017). DnaE consists of the NpuN and NpuC intein fragments. Caging of NpuN and NpuC with truncated fragments of the opposite intein fragment inhibits protein trans-splicing. This allows for the controlled induction of protein _trans_-splicing upon removal of the intein cages (Gramespacher et al., 2017). Here, we utilized NpuN and NpuC to induce linkage of dead Cas9 (dCas9) proteins upon removal of intein cages by cathepsin B cleavage.
3. Assembly and part evolution
The sequence for NpuN was taken from Gramespacher et al. (2017) and optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).
The protein sequence of NpuC1-30 was taken from Gramespacher et al. (2017). Two modifications were made to the sequence of NpuC1-30 by Gramespacher et al. (2017): a repeat of NpuC1-30 was used and three point mutations (K2E, R6E, K11E) were introduced in each repeat. The nucleotide sequence was codon optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.).
4. References
Gramespacher, J. A., Stevens, A. J., nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. https://doi.org/10.1021/jacs.7b02618
Mills, K. V., Johnson, M. A., & Perler, F. B. (2014). Protein Splicing: How Inteins Escape from Precursor Proteins. Journal of Biological Chemistry, 289(21), 14498-14505. https://doi.org/10.1074/jbc.R113.540310
Ventura, B. D., & Mootz, H. D. (2019). Switchable inteins for conditional protein splicing. Biological Chemistry, 400(4), 467-475. https://doi.org/doi:10.1515/hsz-2018-0309
Wang, H., Wang, L., Zhong, B., & Dai, Z. (2022). Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology [Mini Review]. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.810180
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