Parasites were transfected as previously described either by electroporating ring-stage parasites18 or nucleofection of schizont stages11,19. For transfections with circular DNA, 50 µg of each plasmid were used. For transfections with linear DNA, pL7-egfp or pL7-kahrp plasmid was linearized with double-cutting restriction enzyme HincII, extracted by phenol-chloroform, verified for linearization in agarose gel and 10 µg were subsequently used for transfection. Prior to transfection, the appropriate DNA was ethanol-precipitated and resuspended in 10 µl and 30 µl of Tris-EDTA buffer for schizont nucleofection and ring-electroporation, respectively. Lonza Summary: The authors adapted CRISPR-Cas9 expression system to Plasmodium falciparum and transfected it into schizont stages using the Nucleofector 4D technology. This was the first demonstration of CRISPR-Cas9 genome editing in a eukaryotic pathogen. The results show specific gene knockouts and single-nucleotide substitutions achieved in a significantly shorter time than formerly done. The success of this marker-free approach is crucial for consecutive genome manipulations for malaria research and likely for other relevant pathogens.