Molecular mechanisms, functions and applications of RNA-guided DNA transposons

Project Information

BROADCAST

Grant agreement ID: 101115765

DOI

10.3030/101115765

EC signature date 14 September 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 403,29

EU contribution

€ 1 499 403,29

Coordinated by

UNIVERSITAT WIEN
Austria

Periodic Reporting for period 1 - BROADCAST (Molecular mechanisms, functions and applications of RNA-guided DNA transposons)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

The ability to precisely modify genomes has profoundly changed biological research and opened new possibilities in biotechnology and medicine. While CRISPR-based genome editing tools have enabled programmable changes to DNA, a universal strategy for efficient and precise gene targeting and insertion across different organisms and cell types is still lacking. This limitation represents a major bottleneck for many applications, from basic research to gene therapies.

Transposons are natural gene transfer vehicles found in virtually all forms of life. Through their distinctive ability to mobilize DNA within and between genomes, they have played a central role in shaping genome architecture, regulation, and evolution. These same properties make transposons attractive candidates as broadly applicable genome engineering tools. However, despite their ubiquity and biological importance, the molecular mechanisms underlying many transposon systems remain poorly understood, and their technological potential for site-specific DNA manipulation is only beginning to be explored.

The overall goal of the BROADCAST project is to advance our mechanistic knowledge of recently identified RNA-guided DNA transposons in order to understand their biology and guide their repurposing as next-generation genetic technologies. The project focuses on three main directions: first, revealing how CRISPR-associated transposons (CASTs) achieve targeted DNA cut-and-paste reactions (Objective 1); second, studying newly discovered transposon-encoded proteins that act as RNA-guided DNA targeting enzymes, with the ultimate goal of supporting their application as versatile genome editors (Objective 2); and third, using insights into CASTs to develop programmable DNA insertion tools (Objective 3).

By combining fundamental biological research with early translational efforts, BROADCAST aims to advance the molecular understanding of transposition while laying the groundwork for novel genome editing approaches. In the long term, the project’s outcomes may expand the precision and wide applicability of strategies for genetic manipulation, with potential relevance to genome research, biotechnology, and biomedical applications.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Within Objective 1, we investigated the molecular mechanisms of RNA-guided DNA transposition by CRISPR-associated transposons (CASTs), focusing on the Pseudoalteromonas type I-F CAST, currently the most efficient CAST for human genome editing. We reconstituted the activity of the heteromeric TnsA-TnsB transposase in vitro and defined key determinants of nucleoprotein complex assembly, transposon end recognition, and cleavage. Biophysical studies revealed DNA-dependent formation of a paired-end complex through transposase oligomerization, enabling coordinated cleavage of both transposon termini. Cryogenic electron microscopy (cryo-EM) analysis of the paired-end complex revealed the architecture of the transpososome in an immediate post-cleavage state. These studies provided the first structural and mechanistic framework for type I-F CAST transposition, advancing fundamental understanding and informing the continued rational optimization of CAST systems (Objective 3).

Within Objective 3, we investigated key challenges in CAST efficiency and delivery by analyzing hyperactive PseCAST variants generated by directed evolution to define the molecular basis of enhanced activity. In parallel, we identified naturally occurring hybrid CAST systems combining targeting modules with transposition machinery belonging to distinct CAST subtypes, providing a foundation for further optimization of CAST-based gene insertion tools.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project advances the state of the art by establishing a mechanistic framework linking RNA-guided machineries to DNA transposition. By integrating biochemical, structural, and functional analyses, the work provides fundamental insights into how programmable DNA processing systems operate in nature and how they can be harnessed for technology.

Under Objective 1, the project has generated new mechanistic insights into RNA-guided DNA transposition by CASTs, particularly with respect to transposon DNA recognition and excision. Ongoing studies aim to complete the mechanistic description of the transposition pathway by elucidating DNA integration and its regulation. These results address key gaps in our understanding of CRISPR-guided transposons in bacterial genome plasticity and horizontal gene transfer, while directly informing efforts to translate CAST machineries into site-specific gene insertion tools (Objective 3).

Work under Objective 3 builds on these mechanistic insights to address key challenges in CAST efficiency, delivery, and robustness. Ongoing studies of hyperactive transposases and naturally occurring hybrid CAST systems aim to define their molecular properties and functional performance in vitro and in bacteria. By identifying and overcoming molecular constraints, this work enables further optimization and technological advancement of CAST-based tools.