Project
Description

Abstract

The spatial arrangement of DNA sequences is essential for processes such as DNA repair, transcriptional regulation and chromatin remodeling. Despite this importance, recreating proximity-dependent DNA-DNA interactions in vitro and in vivo has proven difficult, hindering both basic research progress and the development of new therapies. In response to this challenge, we introduce PICasSO, the Plasmid Integrated Cas-Stapled Origami system designed to mediate defined DNA-DNA interactions in a site-specific, adaptable, and scalable manner. PICasSO employs CRISPR-based RNA-guided protein stables that are able to precisely and efficiently link custom DNA sequences or genomic loci in a scalable manner. In contrast to conventional DNA nanotechnology, such as DNA origami, PICasSO is fully genetically-encoded and thus applicable in vivo. Moreover, PICasSO can be easily functionalized with additional proteins. We are also developing an innovative DNA delivery system to enable the transfer of our large constructs to mammalian cells for therapeutic applications of PICasSO. This system hijacks bacterial conjugation to transfer large plasmids of up to 100 kilobases to mammalian cells, overcoming the size limits of existing methods thus broadening the range of DNA delivery tools. The modular design of our PICasSO toolbox will empower users to control and manipulate the spatial organization of DNA - from simple plasmids to entire genomes.

Introduction

It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects. Specific DNA folds, DNA-DNA contacts or nanostructures are typically realized in the form of DNA origami, in which short staple sequences bind and fold a long DNA scaffold into a desired shape. While being a powerful method, DNA origami is inherently limited to in vitro production, as its assembly requires denaturing steps at high temperatures (Douglas et al., 2009).

While efforts have been made to use conventional DNA-binding proteins such as transcription activator-like proteins are capable of tightly binding DNA, they only target to their cognate binding motifs rendering them unsuitable to control DNA conformation at scale and in a programmable fashion (Praetorius and Dietz, 2017).

To circumvent these substantial limitations, our project aims to create a versatile tool box for programmed spatial organization and folding of DNA in vivo.

Project outline

Our staple design mainly focuses on dCas9-dCas12a proteins (Wu et al., 2022). Generally speaking, dCas9 and dCas12a recognize specific DNA motifs while being linked via a flexible peptide linker. In order to establish our platform, we are testing different DNA-binding proteins to create different bivalent DNA-binding staples suitable for many use cases.
To investigate the functionality of our staples and their impact on gene expression, we will bind, link and fold plasmids in vitro and in vivo. Additionally, the utilization of Cas12a's endoribonuclease activity for gRNA processing enables expression of multiple staples in one transcript, providing a virtually limitless amount of gRNA sequences and thus highly multiplexed stapling. In order to further modularise our platform, we will establish different stimuli-responsive linker systems. This allows for the precise modulation of staple (dis-)assembly under specific intrinsic or extrinsic conditions. Furthermore, we will also test different established Cas modulating peptides, which will allow for further versatility of our system. We will validate our system across diverse mammalian cell lines and bacterial strains, which ensures robust functionality and proper translation to diverse use cases.

Bioinformatics

To ensure its efficient experimental implementation, we first characterize the behavior of our PICasSO system in silico, evaluate challenges faced in the laboratory and provide a simple tool for future development of experiments. To this end, we also model the behavior of our designed system. This includes the shape of our staples, the forces exerted on our designed structure by the staples to identify stress points and the dynamically changing shape of our designs. Additionally, we will optimize the functionality and stability of our PICasSO staple, focusing on different linkers between the Cas proteins and enhanced guide RNA architectures. Finally, these efforts will be combined in a simple tool that will allow users to easily implement our platform and to design customized plasmids expressing the PICasSO system targeting any combination of sequences of interest.

Conjugation

Various applications of the PICasSO system will require very large plasmid sizes. Therefore, a suitable DNA delivery method is crucial for the successful application in vivo and later testing in living organisms. As established methods are limited to a relatively small insert size, e.g. ~5kb in Adeno-associated virus based vectors, we aim to establish a modular system for the bacterial to mammalian conjugation as a novel method to enable the in vivo delivery of large plasmid sizes of more than 100 kb.

During conventional conjugation, DNA is transferred from one bacterial cell to another by development of a conjugative pilus and expression of a T4 secretion system (T4SS), through which a single strand of a plasmid DNA containing an origin of transfer (oriT) can be delivered to a recipient cell. Although normally restricted to bacteria, inter-kingdom conjugation has been reported between bacteria and plant cells (Zambryski et al., 1989), yeasts (Soltysiak et al., 2019) and mammalian cells (Waters, 2001). The application of in vivo inter-kingdom DNA transfer by conjugation is promising but still limited by very low transfer efficiencies. We make use of the conventional conjugation mechanism by engineering E.coli to carry a helper plasmid encoding the conjugation machinery as well as the customizable transfer plasmid carrying the ori T.

To then optimize the conjugation efficiency, we engineer the donor bacteria to express synthetic adhesins. These are displaying nanobodies that bind to a cell type-specific surface marker of the recipient mammalian cell. Thus, we aim to increase connection stability and proximity between bacteria and mammalian cells as special proximity has been shown to have a major influence on conjugation events (Robledo et al., 2022).

Outlook and Application

The PiCasSO platform is a great addition for many areas of research. It will serve as a foundational research tool, enabling scientists to investigate critical questions that require DNA proximity. It could also be an important addition to synbio systems to programme genome organization in synthetic cells. From fundamental genome studies to assembling plasmids and regulating transcription, our system's modularity will enable versatile applications across various contexts.

We will dynamically modulate gene expression via the folding state of a plasmid. By creating a plasmid system that contains trans-activating domains and a gene of interest, gene expression will be dependent on close spatial proximity of both domains, which will be induced by the binding of our designed staples. This concept can be used in the context of extrachromosomal DNA (ecDNA), which plays a crucial role in the progression of cancer, notably enhancing genetic diversity and oncogene activity through mechanisms such as enhancer hijacking in different cancer types (Shen et al., 2023), (Kim et al., 2020). This process involves relocating enhancers, typically distant within the genome, into close proximity to oncogenes on circular ecDNA structures. The proximity of enhancers to oncogenes leads to their excessive activation, driving rapid and uncontrolled tumor growth while enabling independent gene replication and expression. The stochastic nature of ecDNA formation poses significant challenges for its study and replication in laboratory settings, complicating efforts to fully understand its role in oncogenesis. This randomness, coupled with its prevalence in aggressive cancer types, underlines the urgent need for research focused on understanding the influences of ecDNA on cancer development (Kim et al., 2024). Our platform will allow users to study ecDNA formation events by bringing enhancers and oncogenes into close proximity, and to analyze interactions among multiple ecDNA. Additionally, it will allow for the relocation of ecDNA to study the effects of different cellular locations on the ecDNA activity. We might also be able to create ecDNA hubs and study their cross-activation and inactivation. Our platform represents an initial step with significant potential to enhance our understanding of ecDNA's role in oncogenesis and to set the basis for the development of targeted cancer therapies.
These examples show how the modular design of our system can be used for investigating interactions between multiple genetic elements, even in vivo. This adaptability demonstrates the platform's utility in a broad range of genomic research applications, providing users with the tools needed to explore and manipulate the influences of inducing spatial dsDNA proximity.

References

Chen, Y., Qiu, Q., She, J., and Yu, J. (2023). Extrachromosomal circular DNA in colorectal cancer: biogenesis, function and potential as therapeutic target. 42, 941-951.

Dong, Y., He, Q., Chen, X., Yang, F., He, L., and Zheng, Y. (2023). Extrachromosomal DNA (ecDNA) in cancer: mechanisms, functions, and clinical implications. Front Oncol 13, 1194405. 10.3389/fonc.2023.1194405.

Douglas, S.M., Dietz, H., Liedl, T., Högberg, B., Graf, F., and Shih, W.M. (2009). Self-assembly of DNA into nanoscale three-dimensional shapes. 459, 414-418.

Kim, H., Nguyen, N.-P., Turner, K., Wu, S., Gujar, A.D., Luebeck, J., Liu, J., Deshpande, V., Rajkumar, U., Namburi, S., et al. (2020). Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers. 52, 891-897.

Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J.W., Yoon, M., Rim Shin, H., Kim, J.-S., and Kim, Y. (2017). Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. 8, 1723.

Praetorius, F., and Dietz, H. (2017). Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes. Science 355, eaam5488. doi:10.1126/science.aam5488.

Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L., and de la Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Res 50, 12938-12950. 10.1093/nar/gkac1164.

Soltysiak, M.P.M., Meaney, R.S., Hamadache, S., Janakirama, P., Edgell, D.R., and Karas, B.J. (2019). Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae. Int J Mol Sci 20, 5212.

Watanabe, T., Okada, H., Kanamori, H., Miyazaki, N., Tsujimoto, A., Takada, C., Suzuki, K., Naruse, G., Yoshida, A., Nawa, T., et al. (2020). In situ nuclear DNA methylation in dilated cardiomyopathy: an endomyocardial biopsy study. ESC Heart Fail 7, 493-502. 10.1002/ehf2.12593.

Waters, V.L. (2001). Conjugation between bacterial and mammalian cells. 29, 375-376.

Wu, T., Cao, Y., Liu, Q., Wu, X., Shang, Y., Piao, J., Li, Y., Dong, Y., Liu, D., Wang, H., et al. (2022). Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System. J. Am. Chem. Soc. 144, 6575-6582.

Zambryski, P., Tempe, J., and Schell, J. (1989). Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56, 193-201.