Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9

Area of Science:

• Synthetic biology
• Metabolic engineering
• Molecular biology

Background:

• Introducing complex biosynthetic pathways into host genomes is crucial for producing valuable compounds.
• Existing methods for multigene insertion, like CRISPR-Cas9, are often inefficient and labor-intensive.

Purpose of the Study:

• To develop a standardized, iterative genome editing system for efficient multigene insertion in Escherichia coli.
• To facilitate the construction of microbial cell factories for chemical production.

Main Methods:

• Utilized CRISPR-Cas9 and MetClo assembly based on the Golden Gate standard for modular DNA assembly.
• Developed a toolkit for assembling sgRNA/CRISPR arrays and donor DNAs.
• Optimized multiplex genomic insertion efficiency using strong promoters for tracrRNA expression.

Main Results:

• Achieved up to 100% gene insertion efficiency at a single locus.
• Demonstrated multiplex genomic insertion efficiency of 7.3% with optimized tracrRNA expression.
• Successfully integrated 5-10 genes (5.3–8 Kb) for coenzyme B12 biosynthesis into E. coli.
• Generated 14 antibiotic-free, plasmid-free E. coli producers.
• Developed a recombinant strain yielding 1.49 mg L-1 coenzyme B12, a record titer for E. coli production.

Conclusions:

• The developed genome editing system is user-friendly and versatile for integrating complex biosynthetic pathways.
• This toolbox accelerates pathway engineering and optimization for industrial chemical production.
• The system enables the creation of efficient microbial cell factories without antibiotic or plasmid selection markers.