Fig. 5

Off-targets with only 9 nt of identity to the seed sequence can produce strong fitness defects. a We plot here the proportion of guide RNAs that have an off-target position in a region where guides consistently produce a strong fitness defect. This proportion is shown for guides that target the template strand of non-essential genes but produce an unexpected fitness defect (orange), as well as for guides in the same genes and orientation but that do not produce a fitness defect (blue). This blue curve can be interpreted as the false-positive rate. The green curve is the difference between the green and blue curves. It can be interpreted as an estimate of the proportion of guides whose fitness defect is due to an off-target effect. The maximum is obtained for a perfect match of 9 nt in the seed sequence, which indicates that 9 nt of identity in the seed sequence is enough to produce a strong fitness defect, but mostly false-positive off-target positions are detected when going down to 8 nt of identity. b Fitness effect of guides targeting gene lgt in strain LC-E75. The strong fitness defect produced by the T-bioC guide can be explained by the presence of an off-target position in the promoter of essential gene lgt. c Off-target position of the T-bioC guide in the promoter of lgt essential gene. d Repression of bioC and lgt expression by the T-bioC guide in strains LC-E18 and LC-E75 as measured by RT-qPCR. Points show biological replicates (n = 4), the black bar shows the median