Establishing a transferable prokaryotic prime-editing tool for search-and-replace gene editing

Important results from the project

The project aimed to develop a transferable prokaryotic prime-editing platform for efficient targeted genome-editing in three industrial bacteria. Using a fluorescence-based screening approach, a preliminary tool showed promising results, revealing that gRNA structure, which can influence its degradation and stability, is crucial for editing efficiency. However, other tool components were not tested due to time constraints, requiring further optimisation to enhance the tool´s performance.

Expected long term effects

These findings lay a strong foundation for further optimizing the tool. To fully unlock its potential, additional strategies are needed to enhance gRNA stability and explore other tool components. Moreover, evaluating diverse target sites and mutation types will be essential for comprehensive characterization. Despite the need for further refinement, this tool represents a significant step toward more efficient genome editing in these species, with important implications for biotechnology.

Approach and implementation

The project simultaneously explored the candidate tool in the three selected species using a fluorescence-based screening strategy. Initially, achieving a transferable tool functional in the three species proved challenging, primarily due to the need for different plasmids for tool expression. However, the use of a broad-host range plasmid ultimately enabled tool expression in all species. Subsequent screening was then carried out as planned.