Figure 3.

Inducible expression of Cas9 in M. smegmatis and BCG. E. coli transformed with pCas9 was used as positive control (C+). Total protein extracts of wild-type mycobacterial strains were used as negative controls (C-). (A) Western blot of total protein extracts of M. smegmatis transformed with pKLM-CRISPR vector with either Cas9 expression induced (+) or not induced (-) with tetracycline. (B) Western blot of the expression of Cas9 in BCG induced with tetracycline over time (24, 48, 72, 96 or 120 h). The Western blots were probed with monoclonal anti-Cas9 antibody (1:1,000). Molecular weight markers (MW) are indicated in the left and the expected molecular weight of Cas9 indicated by an arrow (~160 kDa).