Fig. 3
From: Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli
The deletion mechanism of pDG-A-X series during DNA replication. Plasmid pDG-A-X was designed for double gRNA expression: gRNA1 and gRNA2. Each gRNA containing a 20-nt spacer sequence (yellow or green) and an 82-bp scaffold (blue) was transcribed by a constitutive promoter J23119 (purple). During the DNA replication process in E. coli, pDG-A-X series generated the Types I or Type II slipped misalignment of the Okazaki fragment, which formed a loop within the lagging strand template to facilitate the formation of deletion. Deletion Type I: When the second promoter J23119 of the template strand was employed as mispaired position, the deletion of the first gRNA expression cassette occurred, leading to the formation of pDG-A-X-M1. Deletion Type II: When the repeated gRNA scaffold mediated the plasmid recombination, the second gRNA expression cassette of pDG-A-X series was deleted to form pDG-A-X-M2. The golden arrows indicate the direction of plasmid replication