Construction of Inducible CRISPR/Cas9 System

The plasmid pgRNA was assembled using type IIS restriction enzymes‐based assembly method, Golden Gate,^[28] for which DNA primers were designed using J5 Device Editor.^[29] Inducible gRNA plasmids were constructed for guiding CRISPR/Cas9 to the target locus between ddpX and dosP on the chromosome of E. coli MG1655. The backbone of pgRNA was PCR amplified from pACYC184‐M,^[30] and the gRNA with its promoter was amplified from the plasmid pRed_Cas9_ΔpoxB300.^[31] The plasmid pgRNA(N20PAM) was constructed using the same method. Plasmid pRedCas9 for the inducible expression of λ‐Red and Cas9 was modified from pRed_Cas9_ΔpoxB300 and assembled using the Golden Gate method.