a Schematic of plasmids used to compare performance of CRISPR-control (pCRISPR Amp), Pro-AGFP (pPro-AGFPAmp: gRNA2 + GFP within homology arms), and external placement of gRNA2 outside of the homology arms flanked cassette (pgRNA Out-Amp: GFP-only within homology arms, gRNA outside of HA-cassette). b Recovery of E. coli CFU following CRISPR-control or Pro-AG using the three different plasmid configurations indicated in a and the various induction conditions indicated in the key. c Sequence analysis of targeted plasmids. pETg plasmids recovered from CRISPR-control-treated colonies (green circle) all display intact target sequence (“escapers”). Analysis of all Pro-AGFP-recovered pETg plasmids (blue circle) confirmed precise insertion of the gRNA + GFP cassette at the gRNA cut site, which consists of the full gRNA cassette (scaffold, purple; gRNA, pink and black, and tet promoter, gray) plus GFP. Although the gRNAOut-targeted pETg configuration (red circle) resulted in ~ 100-fold less efficient targeting of the bla target gene than the Pro-AGFP configuration, all plasmids isolated from colonies selected on Gm plates carried precise insertions of the GFP-only cassette. Data in b are plotted as the mean ± SEM, representing three independent experiments performed in triplicate and analyzed by Student’s t test. N.S. = not significant (P > 0.05); ***P < 0.001; ****P < 0.0001. Source Data are available in the Source Data file.