A Multiplex Genome Editing Method for Escherichia coli Based on CRISPR-Cas12a

Area of Science:

• Synthetic Biology
• Microbial Genomics
• Genetic Engineering

Background:

• Existing methods for Escherichia coli genome editing are limited in rapidly integrating multiple large DNA sequences into distinct genomic locations.
• A need exists for efficient tools to perform multiplexed gene insertions in bacterial chromosomes.

Purpose of the Study:

• To develop a novel, rapid method for simultaneously inserting multiple heterologous genes into specific loci within the E. coli genome.
• To demonstrate the utility of this method for metabolic engineering and its applicability to other bacterial species.

Main Methods:

• Utilized a CRISPR-Cas12a system for the selective elimination of non-recombinant cells via double-stranded DNA cleavage.
• Employed phage-derived λ-Red recombinases to facilitate homologous recombination between donor DNA and targeted chromosomal sites.
• Developed a two-plasmid system for multiplexed gene insertion without requiring chromosomal markers.

Main Results:

• Successfully achieved the simultaneous insertion of up to three heterologous genes into distinct sites in the E. coli genome in a single round.
• Constructed a high-yield recombinant E. coli strain for the industrial production of 5-aminolevulinic acid (ALA).
• Adapted a similar two-plasmid system for genome editing in the extremophile Halomonas bluephagenesis.

Conclusions:

• The developed CRISPR-Cas12a and λ-Red recombinase-based system enables efficient and rapid multiplexed genome engineering in E. coli.
• This method facilitates the construction of metabolically engineered strains for industrial chemical production.
• The system shows promise for broader application in the genetic manipulation of diverse bacterial species.