FIGURE 1.

Toxoplasma gondii rhoptry organelle proteins (ROP) knockout (KO) mutant construction. (A) Schematic illustration of CRISPR-Cas9-mediated gene KO by insertion of the pyrimethamine-resistant DHFR^∗-Ts in the ROPs coding sequence. (B–E) KO forward and KO reverse primers were used to amplify the small fragment with 30 s extension time (B,C) or the large fragment with 4 min extension time (D,E). Diagnostic PCR demonstrates DHFR^∗-Ts integration and ROP genes disruption in a representative clone compared with wild-type RH strain. (F,G) RT-PCR screening of ROP gene KOs. Small fragment around the insertion site for each ROP gene was not detected by RT-PCR demonstrating that the coding sequences were disrupted.