A double-locus scarless genome editing system in Escherichia coli.

Objective: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette.

Results: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with 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 carrying cas9 and λ 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.