Fig. 2
Functional characterization of NcGRA64(a,b). A Schematic illustration of the disruption of NcGRA64(a,b) by CRISPR/Cas9-mediated homologous gene replacement. B Diagnostic PCRs on two Δncgra64(a,b) clones (1 and 2). (F2-R1) and (F1-R2) check the correct integration of the selection marker to the NcGRA64(a,b) genes locus, whereas (F3-R3) and (F4-R4) examine the deletion of the NcGRA64(a,b) genes. C Plaque assay comparing the growth of parent Nc1 and Δncgra64(a,b) strains in vitro. Purified tachyzoites were used to infect HFF monolayers (200/well) seeded in 12-well plates, and plaques were stained 9 days later. D Intracellular parasite replication of Nc1 and Δncgra64(a,b) strains. Data were compiled from three independent assays, and a total of 200 PVs of each strain were counted in each assay. Data were analyzed using the Chi-square test. E Mouse survival after infection with 5 × 106 and 8 × 106 doses of Nc1 or Δncgra64(a,b) strains. BALB/c mice were infected with tachyzoites from Nc1 or Δncgra64(a,b) strains by intraperitoneal injection, and the survival of the mice was monitored daily. Statistical analysis was performed using the life test in a statistical analysis system (SAS Institute Inc., USA). The data are representative of two experiments with similar outcomes