Applications

Creating Cell Line Models to Investigate TAD Boundary Collapse as a Major Driver of Various Diseases

Topologically associated domains (TADs) boundary disruptions can lead to gene dysregulation causing cancer and developmental disorders. Studying the underlying molecular mechanism remains challenging due to the lack of robust model systems. PICasSO can address this issue by enabling scientists to mimic TAD boundaries and their collapse in a well-controlled manner.

Problem

TADs are the structural and functional units of chromosomes, playing a critical role in regulating gene expression by facilitating interactions between enhancers and promoters via chromatin looping. These regulatory loops operate within the spatial range defined by TADs, insulated by boundary elements consisting primarily of CCCTC-binding factor (CTCF) sites. Together with cohesin rings, CTCFs stabilize TAD boundaries, preventing enhancers from interacting with genes in neighboring domains (see Fig. 1A). (Krijger and Da Laat, 2024)

The dysregulation of TAD boundaries has been implicated in many cancers including acute myeloid leukemia, as well as developmental disorders such as limb malformations (Claringbould and Zaugg, 2021). Disruption of chromatin topology can occur, for instance, through mutations or methylation of CTCF binding sites resulting in the merging of adjacent TADs. This leads to gene dysregulation, as enhancers from one TAD may influence genes in another, potentially driving carcinogenesis and other pathologies (Krijger and Da Laat, 2024; Tettey et al., 2023).
As it is ethically and legally difficult to create model cell lines from patient tissue, we need a system that can accurately replicate TAD boundary collapse in a controlled and inducible manner. Also, to differentiate between correlation and causality in context of observed 3D genome changes, the ability to selectively and directly perturb the genome’s topology is essential to dissect related disease mechanisms and identify suitable and selective therapeutic targets.

How PICasSO Can Help

With PICasSO, we can, for the first time, create inducible stable cell lines that allow precise dissection of TAD boundary collapse and its effects on gene regulation. Such a cell line will enable researchers to observe the consequences of boundary collapse and chromatin reorganization on gene expression and disease.
This can be achieved by combining CRISPR base editing with our multiplexed Cas stapling system. The first step is to use CRISPR base editing to mutate CTCF binding sites in HEK293T cells, causing TAD boundary collapse and allowing interactions between enhancers and promoters from neighboring TADs (see Fig. 1B). Next, the boundary will be restored using Cas stapling, recreating chromatin insulation (see Fig. 1C). Using RNA sequencing (RNA-seq) as a functional read-out, researchers could track transcriptional changes in cells with intact, collapsed, and restored boundaries, offering insights into the mechanistic coupling between chromatin loops and gene expression. Taking the concept one step further, by functionalizing the Cas staple with a cathepsin B cleavable linker , the TAD boundary collapse could be made inducible, providing temporal control. This versatile model will facilitate the study of dynamic gene regulation and help to identify therapeutic targets for diseases including cancer and limb malformation.

Creating Functional, Genomic DNA Interaction Maps

Promiscuous and context-dependent behavior of regulatory sequences like enhancers, along with high false-positive rates in chromatin interaction mapping methods such as Hi-C, make it difficult to establish functional links between spatial DNA rearrangements, gene regulation, and function, hindering (disease) mechanism studies. PICasSO could be employed to address these challenges by mapping DNA interactions and validating their functional relevance with RNA sequencing.

Problem

The human genome contains a vast array of regulatory sequences like enhancers, which often exhibit unpredictable, promiscuous, and context-dependent behavior. More than 90% of disease-associated genetic variants are located within non-coding DNA, especially in presumptive enhancers. A growing body of evidence suggests that various diseases can be caused by faulty regulatory connections between enhancers and their target genes due to a misregulation in genome structure, leading to the dysregulation of gene expression (Krijger and Da Laat, 2024). Understanding these interactions is crucial for unraveling disease mechanisms and developing targeted therapies.
Traditionally, chromatin interactions are mapped using methods like Hi-C and Micro-C, which capture the spatial organization of chromatin in a given cell population source (Kempfer and Pombo, 2019; Lee et al., 2022). While generally effective, these methods expose significant drawbacks, as noted by Prof. Carl Herrmann during an expert interview. One major issue is a high false-positive rate as well as the “correlation vs. causality” dilemma due to the lack of a functional cross-validation. As a result, clear links between observed chromatin interactions and gene regulation are difficult to establish. Furthermore, Hi-C provides a static snapshot of chromatin architecture, failing to account for dynamic changes, for instance throughout the cell cycle

How PICasSO Can Help

PICasSO offers a novel solution to these challenges. Unlike traditional methods, it does not require cell lysis, enabling researchers to recreate chromatin interactions detected by Hi-C and validate their functional relevance using RNA sequencing. That way PiCasSO can be used to confirm interesting DNA-DNA interaction detected by Hi-C with transcriptomics as a functional readout.
PICasSO can even be taken a step further. By combining it with a lentiviral library of fusion guide RNAs and single-cell RNA sequencing, one could significantly enhance the throughput of functional chromatin interaction studies, effectively creating a PICasSO screening platform similar to conventional CRISPR screens (see Fig. 2). With over 10,000 targets already validated using regular CRISPR screens, we expect to achieve similar experimental scales with PICasSO. The resulting data will allow new functional-mechanistic insights into the 3D genome architecture and offer a thus-far unexplored perspective on information encoding in the spatial dimension of eukaryotic genomes.
In our view, integrating Hi-C and PICasSO screens, represents the next important leap in chromatin interaction mapping, combining high-resolution spatial data with functional validation to better understand chromatin architecture and its role in disease.

Uncovering the Role of ecDNA Hubs in Oncogenesis

Extrachromosomal DNA (ecDNA) hubs, prevalent in cancer, drive oncogene amplification and tumor growth - a process that is still poorly understood. PICasSO allows for studying these hubs by enabling their precise manipulation and observing their influence on gene regulation.

Problem

ecDNA is a unique form of DNA found outside the normal chromosomal framework in a cell’s nucleus, predominantly in various cancer types (see Fig. 3A). Unlike linear chromosomal DNA, ecDNA is circular, enabling it to replicate independently of the cell cycle (Yi et al., 2022). ecDNA is frequently formed by end-to-end ligation following the occurrence of DNA double-strand breaks due to the excision of chromosomal fragments. ecDNA plays a crucial role in cancer biology, driving gene amplification and expression of oncogenes like EGFR, MYC, and CCND2 (Yi et al., 2022). One key mechanism that amplifies the oncogenic potential of ecDNA is its clustering into ecDNA hubs. These hubs are “glued” together by bromodomain and extra-terminal domain 4 (BRD4), a protein known to play a role in transcription regulation. Further, they recruit RNA polymerase II (RNAPII) to transcribe genes located on ecDNA or other chromosomal regions. This process enhances the transcriptional activity of oncogenes, thus promoting tumor growth (Weiser et al., 2022).

How PICasSO Can Help

In our interview, PD Dr. Frank Westermann pointed out that manipulating ecDNA hubs has been a persisting challenge in cancer research. One key difficulty lies in inducing proximity between different ecDNAs and observing how these interactions affect transcriptional regulation. This is where the PiCasSO system comes into play. Leveraging its programmable nature, PiCasSO can precisely target the unique sequences formed during the circularization of ecDNA. Using PiCasSO, researchers can engineer cancer cell lines to either promote or disrupt the formation of ecDNA hubs, allowing them to study the downstream effects on gene expression (see Fig. 3B). This approach enables the control of ecDNA clustering, providing insights into how ecDNAs interact within the nucleus. By artificially inducing the formation of hubs, one could analyze the impact on transcriptional activity using RNAseq, revealing how the clustering promotes oncogene expression. Conversely, disrupting these hubs allows researchers to observe the consequences of disrupting transcriptional machinery recruitment, such as BRD4 and RNAPII, potentially offering new strategies for cancer research and therapeutic interventions.
Such control could help uncover the underlying mechanisms by which ecDNA amplifies oncogene transcription. Furthermore, PiCasSO can be applied to move ecDNA hubs to less transcriptionally active regions, such as the basal lamina, reducing their influence on gene expression and potentially slowing cancer progression (Ruegg et al., 1992).
This experimental pipeline offers a novel avenue to explore how altering the spatial dynamics of ecDNA impacts oncogenesis, rendering PiCasSO a promising tool for developing targeted future cancer therapies.

Future Visions

Beyond these immediate use cases of PICasSO, we foresee a variety of exciting avenues to apply our toolbox further into the future. For instance, we envision the use of 3D genome engineering for fine-grained control of cell differentiation as well as to precisely direct cell states in vivo, with numerous biomedical applications. Moreover, we believe that PICasSO could help shape evolutionary trajectories by defining DNA cross-over sections during meiosis, directing homology-directed repair in cells and functionally connecting selected pieces of DNA during cell division as well as horizontal and vertical gene transfer. Finally, we believe that PICasSO even has the power to facilitate the engineering of customized 3D genome architectures entirely from scratch to equip simple cells or fully synthetic cells with sophisticated, computer-designed, dynamic and controllable genome 3D conformations - thereby allowing synthetic biologists to rationally encode information in the spatial dimension of the genome.