Fig. 3: Characterization of the RECKLEEN 1-based method for gene deletions.
A Phenotypic comparison between wild-type (WT) and Δwzi Kp. Deletion of wzi, which encodes an outer membrane protein anchoring the capsular polysaccharide, led to observable changes in colony morphology and size. Morphological differences between WT and wzi-deleted colonies were used for prescreening prior to PCR validation. B Proof-of-concept evaluation of the RECKLEEN method for the deletion of various genes. n = 6 independent biological replicates (three per experiment, across two independent experiments). Individual replicates are shown as circles; filled and open symbols denote independent experiments. Error bars indicate standard deviation from the mean. C Effect of different donor DNA (dDNA) templates on the RECKLEEN editing efficiency. dDNA templates of varying lengths and types (dsDNA: 500 bp/500 bp, 100 bp/100 bp, and ssDNA: 50 bp) were used to assess the method’s efficiency for wzi deletion (by colony morphology), with (+ATc) and without (-ATc) cas9 and sgRNA expression induced. n = 6 independent biological replicates (three per experiment, across two independent experiments). Individual replicates are shown as circles; filled and open symbols denote independent experiments. Error bars indicate standard deviation from the mean. D Killing efficiency for different sgRNAs targeting the sequence of various deletion targets in the Kp genome. EP denotes usage of the empty plasmid (Level 0* plasmid: contains pMB1 origin of replication, aac3(IV) apramycin resistance), Cas9 denotes usage of Ptet Cas9 plasmid (Level 1 plasmid: Ptet cas9), and Ct gRNA indicates usage of the RECKLEEN 1 plasmid with control sgRNA. The sequences of sgRNAs are detailed in Supplementary Table 3. The killing efficiency was calculated as follows: \({Killing\; efficiency}\left[ \% \right]=1-\frac{\frac{{CFU}}{{mL}}{in\; presence\; of\; inducer}\left({with\; counterselection}\right)}{\frac{{CFU}}{{mL}}{in\; absence\; of\; inducer}\left({without\; counterselection}\right)}* 100\) to measure the percentage of killed cells upon induction of the cas9 and sgRNA expression by ATc. Data represents the mean of three biological replicates (n = 3), individual replicates are shown and the error bars represent the standard deviation from the mean.
