Fig 1. Effect of sgRNA strength and drug concentration on abundance of mutants in rpoB in a CRISPRi library treated with RIF (D5).

(A) Comparison of fractional abundances of sgRNAs in rpoB (+ATC / −ATC) to their strengths (in the form of extrapolated LFCs 25 generations in the future). There is a strong correlation of depletion and sgRNA strength in rpoB (RNA polymerase beta subunit, target of rifampicin). There is a linear relationship between these two values, evident by the line of best fit (R² = 0.82). Since strength is measured as extrapolated LFC, the more negative the LFC, the stronger the sgRNA. Here we see that almost linearly, as sgRNA strength increases, abundance decreases. (B) Regression lines for log(relative abundance) against log(concentration) for all sgRNAs in rpoB in a library treated with RIF D5. Although the starting abundance varies, the majority of the regression lines show a negative slope, demonstrating that as concentration of RIF increases, the abundance of sgRNAs in rpoB decrease. The lines that reflect the extremes of the sgRNA strength (orange or blue), are flat and do not show much change in abundance. Comparatively, the middle of sgRNA strength range (navy blue) show the greatest negative slopes reflecting this is the region of ideal sgRNA strength. (C) Comparison of sgRNA strength and slopes of a regression of log(relative abundance) against log(concentration) for each sgRNA in rpoB in a library treated with RIF D5. Each slope (one for each sgRNA) seen in Panel B versus its strength show a parabolic curve. The strongest sgRNAs (left on the plot) and the weakest sgRNAs (right side on the plot) show slopes around 0. These regressions are the flat lines in at the top and the bottom of the graph in Panel B. As seen in Panel A, with strong sgRNAs (left of plot), we already have a low starting abundance, so with increasing concentration, there is little depletion. With weak sgRNAs (right of the plot), starting abundances are high, but the sgRNA is too weak to show depletion with increasing concentration. The minimum of the parabolic curve (dotted line) are sgRNAs of intermediate strength where the ability to detect synergy with the drug is maximized