A multifunctional system for genome editing and large-scale interspecies gene transfer

Area of Science:

• Microbiology
• Synthetic Biology
• Genomics

Background:

• Genome editing technologies are crucial for understanding bacterial functions and engineering novel traits.
• Current methods for large-scale DNA transfer and modification in bacteria are often limited by size, efficiency, and complexity.
• The need for adaptable and efficient tools for bacterial genome engineering is growing.

Purpose of the Study:

• To demonstrate the CRISPR SWAPnDROP system for large-scale in-vivo DNA transfer and genome editing in diverse bacterial species.
• To showcase the modularity and adaptability of CRISPR SWAPnDROP for various genome engineering applications.
• To evaluate the efficiency and capabilities of CRISPR SWAPnDROP in model organisms and pathogens.

Main Methods:

• Implementation of the CRISPR SWAPnDROP system in Escherichia coli, Vibrio natriegens, and Dickeya dadantii.
• Demonstration of excision, transfer, and integration of large chromosomal DNA regions between species.
• Utilizing a multi-color scarless co-selection system for improved editing efficiency and quality control.

Main Results:

• Successful large-scale in-vivo DNA transfer and integration between E. coli, V. natriegens, and D. dadantii without size limitations.
• Demonstration of scarless, marker-free, iterative, and parallel genome editing capabilities.
• Validation of the modular platform for DNA library applications and part recycling.
• Significant improvement in editing efficiency and visual quality control via the co-selection system.

Conclusions:

• CRISPR SWAPnDROP significantly expands the scope of bacterial genome editing for large DNA fragment transfer.
• The system's modularity and efficiency make it a versatile tool for diverse applications in synthetic biology and microbiology.
• CRISPR SWAPnDROP offers a powerful, adaptable, and visually controlled approach for bacterial genetic engineering.