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.

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

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.
The protein sequence of NpuC_1-30 was taken from Gramespacher et al. (2017). Two modifications were made to the sequence of NpuC_1-30 by Gramespacher et al. (2017): a repeat of NpuC_1-30 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.

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