Fig. 3

Validation of 30 bp deletions on 78 genes. E. coli BL21(DE3)ΔompA(pCasRed) or E. coli BL21(DE3)(pCasRed) were transformed with different mixtures of pCRISPR-SacB-gDNAs and 70-base dDNAs (either ssDNA or dsDNA) to mediate 30 bp deletion at one of the 78 selected gene loci (y axis). Ten colonies from each transformation were analyzed by PCR to identify those carrying the deletion. X axis indicates the percentage of mutants identified out of the total number of colonies analyzed. Bar height indicates the mutation frequency, while the presence of flat colored squares above gene names in each graph indicates that no mutants were identified out of ten colonies analyzed. Absence of bars or flat colored squares above gene names indicate that the gene mutation was not attempted for those specific genes in the experiment indicated in each bar graph. Red bars/squares indicate mutation experiments using Ld-ss-dDNAs; Blue bars/squares indicate mutation experiments using Lg-ss-dDNAs; Green bars/squares indicate mutation experiments using ds-dDNAs; bars with green downward diagonals indicate mutation experiments with ds-dDNAs in BL21(DE3)(pCasRed). a Bar graph representing mutation success rate using 46 Ld-ss-dDNAs (red bars/squares) and 32 Lg-ss-dDNAs (blue bars/squares). b Gene mutations that failed using the Ld-ss-DNAs (26 genes out of 46) and the Lg-ss-DNAs (4 genes out of 32) were re-attempted using ss-dDNAs targeting the opposite strands. The chart represents the mutation success rate of this second round of experiments. c The bar graph represents the mutation success rate in BL21(DE3)ΔompA(pCasRed) (green bars) and in BL21(DE3)(pCasRed) (green downward diagonal bars) using ds-dDNAs