Figure 2.

CRISPRi of pknB in M. tuberculosis at serial time points. (A) Position of target sequences, shown by purple vertical lines. Numbering refers to the first nucleotide in the target sequence relative to the annotated start of the pknB coding sequence. (B, E, H, K) Expression of dcas9 with and without induction of dcas9 and the sgRNA indicated. (C, F, I, L) Expression of pknB with and without induction of dcas9 and the sgRNA indicated. (D, G, J, M) Growth curves of culture with and without induction of dcas9 and the sgRNA indicated. (N, O) Extended growth curves. (P) Immunoblot of total protein from the last two time points shown in panel (O) using rabbit polyclonal anti-PknB antibody. VC, vector control. The significance of the difference in gene-specific RNA between samples from induced and uninduced cultures is indicated by asterisks above the bar for the induced sample (*P < 0.01; **P < 0.001; ***P < 0.0001).