CASTedit · Repurposing the CRISPR-associated transposons (CAST) system for gene editing applications

The rapid advancements in CRISPR-Cas technology have revolutionized gene editing, yet significant challenges persist, particularly the reliance on DNA Damage Response (DDR) pathways and the limitations in integrating large DNA segments. These constraints hinder the application of CRISPR-Cas systems, especially in non-dividing cells and complex genomic modifications. The recently discovered CRISPR-associated transposon (CAST) systems offer a transformative solution, enabling the insertion of large DNA cargos (10–30 kb) into specific genomic locations without inducing double-strand breaks (DSBs), thereby providing a safer and more versatile genome-editing tool. The CASTedit project aims to repurpose CAST systems as next-generation genome editing tools, with a focus on mammalian cells. Specific objectives include the development of engineered CAST systems (Types V-K and I-D) for precise DNA integration, the exploration of novel CAST complexes for RNA-guided transposition, and their validation at the organismal level. Successful implementation of these systems in eukaryotic cells could overcome current CRISPR limitations, paving the way for innovative applications in cell engineering, regenerative medicine, and biotechnology. Under the guidance of Prof. Guillermo Montoya, a leading expert in the structure-function analysis of nucleases, and with his pioneering research in CAST complex assembly at the University of Copenhagen, I will be well-positioned to achieve the goals of this project. I will leverage my expertise in CRISPR-based genome editing, protein expression systems in eukaryotes, and high-throughput mutant screening to optimize CAST systems for precise DNA integration in mammalian cells. The successful execution of CASTedit will establish me as a prominent researcher in genome engineering, utilizing the host institution’s state-of-the-art facilities and collaborative environment to drive innovation in therapeutic applications.