A double-locus scarless genome editing system in Escherichia coli

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 and λ Red recombination systems are powerful tools for bacterial genome engineering.
• Simultaneous modification of multiple genomic loci remains a challenge for high-throughput applications.

Purpose of the Study:

• To develop a convenient, scarless, double-locus genome editing system in Escherichia coli.
• To leverage CRISPR/Cas9 and λ Red recombination for multiplex genome engineering.

Main Methods:

• A two-plasmid system was constructed, combining Streptococcus pyogenes CRISPR/Cas9 with λ Red recombination genes (gam, bet, exo).
• A second plasmid expressed two guide RNAs (gRNAs) for simultaneous targeting.
• Recombination efficiency was assessed using single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) with varying homology arm lengths (30 or 40 bp) at the galK, lacZ, and dbpA loci.

Main Results:

• Concurrent double-locus recombination efficiencies reached up to 88% for point mutations and 39.7% for deletion/insertion mutations.
• Efficiency varied based on DNA type (ssDNA/dsDNA) and homology arm length.
• High plasmid curing efficiencies were observed: 96% for the gRNA plasmid and 92% for the Cas9/λ Red plasmid.

Conclusions:

• The developed system offers a convenient and efficient method for simultaneous double-locus genome editing in E. coli.
• This tool is well-suited for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.