Objective To develop a convenient double-locus scarless genome editing system inEscherichia coli, based on the type IIStreptococcus pyogenesCRISPR/Cas9 and lambda Red recombination cassette. Results A two-plasmid genome editing system was constructed. The large-sized plasmid harbors thecas9 and lambda Red recombination genes (gam,bet, andexo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting thegalK,lacZ, anddbpA genes inE. coliwith ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 +/- 5.5% (point mutation), 39.7 +/- 4.3% (deletion/insertion), and 57.8 +/- 3.4%-58.5 +/- 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 +/- 3% in 4 h) than the help plasmid carryingcas9 and lambda Red (92 +/- 2% in 9 h). Conclusions The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.