(With Cas9). "Whereas controls using analogous plasmids targeting other regions generated viable parasites, no parasites harbouring a truncated version of PfGNA1 could be obtained (Fig. 7A). This suggests that the PfGNA1 gene plays an essential role for the survival of the parasite. In a second set of experiments, we were also unable to introduce nonsense mutations in the N-terminus of the GNAT domain conserved region. In contrast, in three independent biological replicates carried out in parallel, we successfully managed to obtain viable parasites harbouring synonymous nucleotide substitutions in the same region of the GNAT conserved domain (Fig. 7C). Therefore, nucleotides in the catalytic GNAT domain of PfGNA1 are genetically modifiable as long as the open reading frame is not altered. These data strongly suggest that an unaltered version of the PfGNA1 protein is required for parasite viability, at least during P. falciparum asexual blood stages."