Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genome Engineering

Background:

• Existing genome editing methods have limitations for dynamic and simultaneous mutagenesis in prokaryotes.
• There is a need for versatile tools to modify multiple genomic loci efficiently.

Purpose of the Study:

• To develop and optimize a novel multiplex genome editing system for prokaryotes.
• To demonstrate the system's utility in generating variant libraries and continuous protein evolution.

Main Methods:

• Development and optimization of the retron-mediated genome editing system (REGES).
• Application of REGES for single-, double-, triple-, and quadruple-locus genome editing.
• Generation of pooled and barcoded variant libraries using degenerate RBS sequences.
• Demonstration of continuous in vivo protein evolution by combining retron, base editing, and error-prone transcription.

Main Results:

• Achieved high editing efficiencies: ~100% (single), 85% (double), 69% (triple), and 25% (quadruple) locus editing.
• Successfully generated variant libraries to fine-tune gene expression for improved ethanol tolerance and biotin biosynthesis.
• Demonstrated REGES for continuous in vivo protein evolution.

Conclusions:

• REGES is a powerful and versatile tool for multiplex genome editing in prokaryotes.
• The system facilitates the generation of variant libraries and continuous evolution for synthetic biology and metabolic engineering applications.
• REGES offers broad applicability for advancing genetic engineering in microbial systems.