(a)
(b)
(c)
(d)
Figure 1: Constitutive expression of Cas9 in L. donovani Bob strain. (a) Proteins were extracted from LdBob (LdB) or LdBob expressing Cas9-FLAG (LdB pTB007, 162 kDa) promastigote in logarithmic phase. Twenty micrograms was analysed by Western blotting using the anti-FLAG M2 antibody (right panel). The Coomassie-stained membrane of the blot is included as a loading control (left panel). Protein weight in kDa is indicated on the left. (b) Logarithmic phase promastigotes were seeded at promastigotes/mL and cultured for 8 days. Samples were collected every 24 h to assess cell number (black symbol) and percentage of cell death (white symbol) by flow cytometry in triplicate in two independent experiments. Cell lines: LdB (square) and LdB pTB007 (circle). (c) Proteins were extracted from LdBob or LdBob expressing Cas9-FLAG (LdB pTB007, 162 kDa) axenic amastigotes (48 h after temperature and pH shift) and processed as described in (a). (d) Logarithmic phase promastigotes were seeded at promastigotes/mL, shifted to 37°C and pH 5.5 and cultured for 5 days. Samples were collected and treated as described in (c).