Project
Description

Introduction

All cells in our body carry the same genetic sequence, yet they have highly diverging and specialized functions. Even more, species that look and behave profoundly differently like humans and chimpanzees share almost identical genome sequences (Yang et al., 2019; Mikkelsen et al., 2005).

The answer to this apparent contradiction lies, at least in parts, in another dimension used by living systems for information encoding: The 3D genome conformation. In fact, cells can interpret identical DNA sequences in various ways depending on their spatial genome organization (Rajarajan et al., 2016; Zheng & Xie, 2019).

Over the last decades, synthetic biology has made great advancements in engineering the first dimension of genomes - the DNA sequence. As seen everywhere in iGEM and beyond, we can nowadays easily introduce entirely new genes into organisms, exchange single or multiple nucleotides in existing genes, and control gene activity by implementing or altering regulatory sequences such as promoters or ribosome binding sites.

However, no effective molecular tool exists to date that enables actively shaping the 3D architecture of genomes - despite its profound impact on cell phenotype, health, and even species differences (Rajarajan et al., 2016; Zheng & Xie, 2019).

We, the iGEM Team Heidelberg 2024, share the vision of making a new level of information encoding accessible to synthetic biology by engineering the genome in three dimensions. With our PICasSO toolbox, we provide a foundational advance to both understanding and controlling the code of life.

PICasSO: Engineering the Genome’s Spatial Architecture

Plasmid-Integrated Cas-Stapled Origami (PICasSO) is our pioneering, well-characterized toolbox, designed to rearrange genome 3D architecture with unprecedented spatial precision. At its heart, PICasSO builds on a unique, bivalent CRISPR dead Cas (dCas) complex, composed of a catalytically inactive dCas9 and dCas12a protein, delivered on a single plasmid. The complex is formed by connecting dCas-proteins via our custom-engineered fusion guide RNAs (fgRNA) — chimeras of the dCas9- and dCas12a-specific gRNAs (Kweon et al., 2017). We termed this whole tripartite CRISPR-Cas complex the Cas staple (see Fig. 1). In a Cas staple, each of the dCas-proteins can bind a DNA strand, bringing them into spatial proximity guided by the fgRNA. As the determining factor, the fgRNA defines this DNA-DNA connection's exact distance and flexibility, while being easily programmable and cheap to produce.

Figure 1: The components of a Cas Staple. Upon binding a fusion guide RNA, our custom bivalent CRISPR dCas complex brings two DNA strands into spatial proximity.

But how can such a compact system control the functionality of the 3D genome architecture? In eukaryotic cells, cis- and trans-gene regulatory elements like enhancers and silencers fine-tune gene expression or even render entire gene sets accessible or inaccessible (Halfon, 2020; Riethoven, 2010). Normally, the activity of such regulatory elements is controlled by mediator or insulator proteins within topologically associated domains (TADs) shaping the 3D genome structure (Dixon et al., 2012). PICasSO mimics the function of a mediator but is entirely programmable to any custom DNA locus pair via the underlying fgRNA. By “stapling” an enhancer element into close spatial proximity to the promoter, for instance, PICasSO can be applied to induce and control expression from endogenous genes without the need to add synthetic transcriptional activators. Alternatively, a Cas staple can also block enhancer access to the TATA box of a promoter, creating an insulator effect to prevent gene expression. Beyond mimicking natural systems, PICasSO also enables the engineering of entirely novel regulatory genome architectures by bringing artificial regulatory genetic elements to precise locations within the genome. This synthetic control modality facilitates fine-tuned gene expression control as well as modeling disease mechanisms rooted in disturbed genome 3D organization, e.g. in cancer (see Fig. 2).

Figure 2: Recreating natural 3D control expression by Insulator/Mediator Mimicking and Synthetic Activation. A) Chromosomes are organized into topologically associated domains (TADs), where proximity of enhancers and promoters regulates gene expression. In the same way, PICasSO facilitates artificial genome regulation by controlling chromatin loop formation. We propose three basic modes in which PICasSO can operate: B) Insulator Mimicry and C) Mediator Mimicry, both utilizing endogenous regulatory sequences, as well as D) Synthetic Enhancer Hijacking facilitated by adding an exogenous DNA element encoding a (synthetic) enhancer.

Next to inducing proximity between cis- and trans-acting loci, the PICasSO system can in fact be used for even more complex tasks. Our unique fgRNA design facilitates the use of multiple Cas staples at once, allowing multiplexing. By introducing the dCas9/dCas12a protein encoding genes just once, we can thus mediate spatial connections between multiple, selected loci-pairs simply by supplying several fgRNAs. This is possible because each fgRNA simultaneously encodes two precisely defined target DNA locations to be tethered (see Fig. 3).

Figure 3: Basic principle of the fgRNA design. dCas12a and dCas9 binding to the fgRNA result in a CRISPR Cas staple, which induces spatial proximity between defined pairs of DNA loci in cis or trans. Importantly, Cas staples can be easily multiplexed simply by co-supplying several fgRNAs.

Due to this novel fgRNA design, multiple Cas staples do not interfere with one another and can hence work in an orchestrated fashion to establish complex regulatory networks (see our related DaVinci models for multiplexed DNA stapling). PICasSO’s multiplexing capability enables large-scale, complex spatial rearrangements of functional DNA elements within the 3D genome, opening new doors for synthetic biology, gene therapy, and beyond.

Introducing PICasSO’s Digital Twin: DaVinci

3D genome engineering is a very complex task. To optimize our initial Cas staple design and experimental setups before performing costly and labor-intensive lab experiments, we developed DaVinci — the Digital Architecture for Visualization and Integration of Novel Interactions — a digital twin of our PICasSO toolbox/experiments.

Our model was created in several iterations, each highly benefiting from the input of experts and stakeholders in the field, and offering detailed information on every component of the PICasSO system. To bridge large-scale DNA simulations with atom-level interactions, we established the respective mathematical calculations, combining AlphaFold3, GROMACS, and oxDNA into a single pipeline (Abramson et al., 2024; Berendsen et al., 1995; Lindahl et al., 2001; Šulc et al., 2012). This way, we can dynamically assess protein-DNA interactions at both macroscopic and atomic levels.

DaVinci allows researchers to input parameters such as target sequences, temperature, and salt concentration to predict the feasibility of stapling at specific genomic locations and under custom experimental conditions. Validated with our own experimental data as well as additional data from the literature, DaVinci has significantly accelerated the design and optimization of our PICasSO staples, enabling real-world applications.

Applying PICasSO to Engineering Genome 3D Conformation

We didn’t just stop at creating PICasSO and DaVinci; we were determined to explore their full potential and discover the exciting new research avenues and applications opened by our toolbox and related models. In close exchange and collaboration with leading experts in the field, we identified various promising applications to push the boundaries of PICasSO in real-world science. As one of the most exciting challenges, we tackled enhancer hijacking (see above), a widespread phenomenon in cancer (Wang & Yue, 2024). It describes the increased activation of an (onco-)gene through a distal enhancer brought into spatial proximity due to abnormal 3D structural rearrangements in the genome. This mechanism is a key driver in many malignancies including one of its deadliest forms, Glioblastoma (Yi et al., 2022). Despite its fundamentality in many diseases (Mortenson et al., 2024), enhancer hijacking has been notoriously difficult to study due to the lack of efficient tools to manipulate enhancer-promoter spatial proximity in living cells (J. H. Kim et al., 2019; M.-S. Kim & Kini, 2017)—until now.

Using the DaVinci model to design specialized Cas staples, we successfully engineered synthetic enhancer hijacking and validated it in HEK293T cells.

This marks the first time enhancer hijacking has been experimentally modeled with an approach that is highly modular and can easily be multiplexed. This paves the way for deeper exploration of complex disease mechanisms related to the genome architecture, such as the spatial genome rearrangements observed in Glioblastomas and other aggressive cancers (Yi et al., 2022), and provides a foundation for research on many other pathophysiological conditions.

Given the success of our initial application, we believe that the future possibilities for PICasSO are limitless. Our technology will shed new light on information encoding in cells and could provide new perspectives and insights into genome evolution and cell differentiation.

Moreover, PICasSO has the potential to fuel the next generation of precision therapies, targeting diseases through induced genome reorganization. We believe our standardized PICasSO toolbox, composed of well-characterized parts, guidelines, experimental protocols and mathematical models, will inspire iGEMers and scientists to explore the untapped potential of engineering the 3D genetic code.

For more on our future experiments and applications, see our Applications page.

Expanding PICasSO’s Functionality

To ensure the broad applicability of our system, we developed a comprehensive part collection, with constructs to create and use custom Cas staples of varying complexity and corresponding assays for their standardized experimental characterization. Our toolbox also contains well-characterized, simpler non-Cas staples that serve as a baseline as well as positive control constructs, i.e. benchmarks, providing future researchers with a foundation to establish our assay framework in various cellular contexts as well as laboratory environments. This standardized testing system facilitates the characterization of new, custom protein staple constructs regarding their DNA binding properties and resulting spatial DNA (re-)organization. Central to our toolbox is a newly established EMSA workflow applicable for calculating binding affinities of purified staple constructs as well as a complementary FRET-based assay system for monitoring of DNA-DNA interactions directly in vivo (Hochreiter et al., 2019; McCullock et al., 2020).

As already mentioned in the name of our toolbox, Plasmid-Integrated Cas Stapled Origami, we aim to deliver all parts of a Cas staple on a single plasmid allowing us to easily deliver PICasSO to target cells. As our system is quite complex, this causes a large plasmid size (up to 15 kb) rendering delivery to mammalian cells using conventional transfection or transduction systems a challenge. To overcome this limitation, we conceived and are in the development of a cheap and scalable delivery system for large DNA constructs based on bacterial-to-mammalian interkingdom conjugation. Notably, cell targeting in this new system is enhanced with a target-seeking nanobody, allowing for selective delivery to specific cell types.

The parts in our toolbox are extensively tested, well-documented in the parts registry and accompanying protocols and guides support researchers at every stage of the engineering cycle while creating and using their own Cas staples for engineering cellular genomes in 3D (see Fig. 4).

Figure 4: The PICasSO Engineering cycle: using our part collection to engineer new staples.

Enabling Future Scientists: Education in Synthetic Biology

History has shown that even the most groundbreaking ideas can only succeed when society is open-minded and well-informed. Through conducting a nationwide survey with over 800 participants, we observed that a person’s level of education significantly influences their attitude toward emerging technologies. We recognized education as a crucial element, forming the foundation upon which all scientific advances rely. With our comprehensive educational program, we equip the next generation of scientists with skills to shape the future of synthetic biology.

Our program is built on four key principles:

Dialogue:
We nurture communication inside the scientific community and points of contact with the general public.

Lasting impact:
Our projects persist past iGEM. Through collaborations, we will continue our actions beyond the end of our project and provide an extensive record of all our materials online, freely available to teachers, tutors, professors, and anyone curious.

Holistic approach:
In our educational programs, we offer a broad range of access points to science and synthetic biology. We made sure to address multiple age groups, including theoretical and practical activities, expanding unrestricted educational opportunities.

Scientific foundation:
To ensure our actions are grounded in a robust scientific framework, we collaborated with renowned institutions including the German Ministry of Education, and the German Cancer Research Center as well as teachers, professors, and education experts, creating a high-quality program that meets the demands of modern science education.
To bring these principles to life, our program consists of six complementary projects that come together like pieces of a puzzle. These include a fully funded summer school as well as workshops for school students, complemented by two lecture series covering topics from cutting-edge research to inclusivity.
By fostering a culture of curiosity, dialogue, and responsible innovation, our education initiatives provide the next generation with the tools they need to transform synthetic biology and drive meaningful societal change in the years to come.

Summary

We successfully developed and characterized the PICasSO toolbox and its digital twin DaVinci, offering a transformative solution for achieving precise control over the 3D spatial organization of the genome. This innovation fills a critical gap in genome regulation, allowing programmable manipulation of DNA architecture - a level of information encoding distinct from the DNA sequence - to control gene expression and function. Complemented by an advanced assay system for engineering and testing new staples, PICasSO uses a novel bivalent CRISPR complex and fusion guide RNA to mimic natural mechanisms of DNA-DNA interaction or even create entirely new regulatory networks via synthetic interactions that do not exist in nature. To streamline the engineering process, our sophisticated and validated computer model DaVinci facilitates the creation of new PICasSO designs. Together with our part collection, these tools provide fellow iGEMers and scientists with a tested and well-documented system to explore new science with this foundational technology. Calling on expert interviews we identified key research questions that PICasSO can address and applied our novel Cas staples to model enhancer hijacking, a barely studied yet important driver of cancer progression. In doing so, we demonstrated that PICasSO is working successfully.

With our comprehensive part collection, advanced modeling tools, and public engagement efforts, PICasSO provides a foundational advance to genome engineering in synthetic biology.