Fig 1.
Tandem repeat assisted genome editing (TRAGE) in comparison with the four reported strategies.
(a) Cre/loxP or Flp/FRT systems. (b) The Pop-in/Pop-out strategy. (c) SceI assisted method. (d) Custom endonucleases assisted gnome editing method. (e) A model for the current understanding of how RecBCD responds to DSBs in front of the replication fork. (f) The model for TRAGE: First, desired DNA fragment with the selectable marker flanked by tandem repeats was introduced into the target site via intermolecular homologous recombination assisted by Red enzymes. Then, seamless excision of the selectable marker was realized via DSB repair based on intramolecular homologous recombination among the tandem repeats. The excision of selection cassette was realized via replication fork reactivation by the mechanism shown in (e)
Table 1.
Primers used for genome editing and subsequent testing in this study.
Table 2.
Strain used and constructed in this study.
Fig 2.
Genome editing strategies with TRAGE in Escherichia coli.
(a) Gene deletion. (b) Gene substitution. (c) Gene insertion.
Fig 3.
Manipulation results with TRAGE in E. coli.
(a) PCR analysis of seamless deletion of three genes, substitution of one gene and insertion of one gene (b) Fatty alcohol production by strain with integration of FAR. Values are the mean of three biological replicates ± standard deviation (n = 3), (***) p < 0.001, one-way ANOVA.
Fig 4.
Procedure optimization for TRAGE.
(a) Recombination efficiency before and after optimization. The recombination efficiencies were increased from around 1 to 8%. (b) Recommended procedure for the excision of cat-sacB by TRAGE with moderate efficiency (about 5%) and short time (around 48 h).
Table 3.
Effects of different procedures on the recombination efficiency (%).
Table 4.
Recombination efficiency with the recommended protocol.