Fig. 4

Specific 5 nt seed sequences produce strong fitness defects that can be alleviated by reducing dCas9 concentration. a Distribution of the fitness effect of guide RNAs that share specific 5 nt seed sequences as compared with the distribution of all the guides in the library targeting the template strand of genes. b Plasmid psgRNA was programmed with various guide RNAs sharing either the TGGAA seed sequence or the ACCCA seed sequence and introduced in strain LC-E18. Guides named R1 and R2 have sequences that do not match any position in the chromosome of E. coli. Cells were grown overnight, serially diluted, and plated on LB agar with 1 nM of aTc. Representative figure from four independent experiments. c Distribution of the fitness effect of guide RNAs in our library depending on target gene essentiality and target strand in strain LC-E75, which expresses dCas9 at a lower concentration than strain LC-E18. d Distribution of the fitness effect of guide RNAs sharing the ACCCA or TGGAA seed sequences in strain LC-E18 and LC-E75. e Average fitness effect of guides sharing specific 5 bp seed sequences in strain LC-E18 (x axis) and LC-E75 (y axis). The red line shows a linear regression (slope = 0.2, R² = 0.47). f Fitness effect of guides targeting gene lpoB and gene hisI in strain LC-E75. The strong fitness defect produced by C-lpoB2 can be explained by the presence of an off-target position in the def essential gene