Fig. 2: Quantitative characterization of NT-CRISPR and killing efficiencies with different gRNAs.

a Different morphologies of wild type and ∆wbfF colonies. Image acquired with transmission light. b NT-CRISPR with different amounts of tDNA, targeting wbfF. This experiment was performed with the indicated amount of tDNA with symmetrical 3 kb flanks upstream and downstream of the target sequence. Red and blue bars show results for samples with and without CRISPR-based counterselection, respectively. n = 4 replicates, representing two independent biological replicates (circle or triangle) and two independent experiments (filled or open symbols). Underlying colony counts are provided in Supplementary Fig. S2a and b. c NT-CRISPR with different tDNA fragment lenghts, targeting wbfF. Red and blue bars show results for samples with and without CRISPR-based counterselection, respectively. This experiment was performed with 100 ng of tDNA and the respective fragment length. With the exception of the last bar, tDNA fragments are symmetrical with the same length for the upstream and downstream homologous sequence. n = 4 replicates, representing two independent biological replicates (circle or triangle) and two independent experiments (filled or open symbols). Underlying colony counts are provided in Supplementary Fig. S2c,d. d Results of killing assay for different gRNAs. Killing efficiency is calculated as follows: \({{{{{\mathrm{Killing}}}}}}\,{{{{{\rm{efficiency}}}}}}[ \% ]=1-\frac{{{{{{\rm{CFU}}}}}}/\mu {{{{{\rm{L}}}}}}\,{{{{{\rm{with}}}}}}\,{{{{{\rm{counterselection}}}}}}}{{{{{{\rm{CFU}}}}}}/\mu {{{{{\rm{L}}}}}}\,{{{{{\rm{without}}}}}}\,{{{{{\rm{counterselection}}}}}}}\,\ast 100\). n = 4 replicates, representing two independent biological replicates (circle or triangle) and two independent experiments (filled or open symbols). b–d Bars show the mean of all replicates and error bars indicate standard deviation of the mean. The dashed line indicates the highest possible value. e Calculation of apparent editing efficiency from initial editing efficieny. Initial editing efficiency describes a theoretical value, assuming no enrichment through counterselection against non-modified cells. Apparent editing efficiency provides the expected fraction of correct colonies after counterselection. The red and blue curves use the highest (malQ) and lowest (vnp2) killing efficiencies observed in Fig. 2d.