Fig. 3.

Deletion and mutant phenotype of endogenous cdpk3 gene in the PyCas9ki parasite

A. Schematic diagram of pYCs construct for deleting the endogenous cdpk3 gene. The site for designed sgRNA recognition was indicated as red thunderbolt.

B. Schematic of sgRNA expressing cassettes driven by P. yoelii U6 snRNA promoter in the pYCs plasmid. The protospacer sequences of sgRNA1 and sgRNA2 are indicated as light orange box. sgRNA transcripts were detected by RT-PCR using specific primer pair (p19/p21 and p20/p21) listed in Table S1. Endogenous U6 snRNA transcript serves as an internal control.

C. PCR analysis of genomic DNA from the 17XNL, PyCas9ki, and PyCas9ki parasites transfected with different sgRNA plasmids indicted. Successful integration of left and right homologous arms was detected at specific sites in the parasite directed by both sgRNA1 and sgRNA2, but not control sgRNA targeting irrelevant sequences.

D. PCR analysis of two clones with cdpk3 deletion in the PyCas9ki parasite.

E. In vitro ookinete differentiation of the parental PyCas9ki and PyCas9kiΔcdpk3 parasites.

F. Ookinete gliding motility of the parental PyCas9ki and PyCas9kiΔcdpk3 parasite using Matrigel motility assay. At least 20 cultured ookinetes were measured over a 20 min period. Results are representative of two independent experiments.