Extended Data Fig. 3: Genetic validation of CRISPR/Cas9 modified parasite lines.
From: Babesia divergens host cell egress is mediated by essential and druggable kinases and proteases
(A) Schematic of the GFP reporter plasmid. (B) Schematic of the CRISPR/Cas9 plasmid with a repair template (PKG HR). Detailed repair template schematics can be found in Figs. 3a and 5a. (C) Live cell imaging of B. divergens parasites expressing GFP. (D) RFLP analysis of the uncloned parasite population after transfection with either a plasmid containing a 500 bp repair template (PKG-T651Q) or with a HA-DD-glmS tag flanked by two 500 bp HR regions targeting PKG (corresponding to diagrams in Figs. 3a and 5a including primer positions). (E) PCR and agarose gel electrophoresis to confirm integration of the knockdown tag in each line. The amplified region spans the 3’ tag, with primers outside of the HR (primers are shown in Fig. 3a). The increased size of the PCR product in the transgenic line corresponds to the correctly integrated tag. The expected band sizes for WT and integrated, respectively, for each gene are: PKAc2 (1.1 and 1.6 kb), ASP2 (1.1 and 1.6 kb), ASP3 (1.2 and 1.7 kb), DPAP1 (1.1 and 1.6 kb), CDPK4 (1.6 and 2.1 kb), CDPK7 (1.8 and 2.3 kb), PKG T651Q-glmS/ PKG-glmS (1.1 and 1.3 kb) and PKG-DD-glmS (1.1 and 1.6 kb). (F) Western blot analysis showing knockdown by combined DD (0 mM Shld1) and glmS induction (1 mM GlcN). ASP2 and ASP3 knockdown were induced for 6 h due to growth defects after longer periods. DPAP1 and CDPK4 knockdown were induced for 48 h. (G) Growth of knockdown parasites over 72 h. Each point represents the mean of an individual experiment performed in technical triplicate and n = 3. The mean ± SD is shown. Data is normalized so +Shld is 100% for each replicate. No statistical differences are observed.
