We observed decreased viability in response to transfection of the SAG1-targetting plasmid, although the loss was more pronounced in DKU80 parasites, which exhibited a 96% decrease, compared to the 78% decrease found in wild-type parasites. In both strains, viability was improved in the absence of a targeting protospacer, and further improved in the absence of the nuclease. However, even in the absence of Cas9, the DKU80 strain showed a significant decrease in viability, which we cannot attribute to the effects of SR 3576 CRISPR/Cas9. This effect may be caused by homology of the control plasmids to the genome or a more general response to foreign DNA in the DKU80 strain, although the relatively low rates of homologous recombination in the absence of antibiotic selection do not agree with the significant decrease in viability we report. The adverse effect of plasmid transfection and the predicted susceptibility of the DKU80 strain to double-stranded breaks both contribute to the decrease in gene disruption frequency observed in the absence of NHEJ. It is not surprising that double-stranded DNA breaks are also detrimental to the wild-type strain, given that not all cells will be able to repair the lesions despite the efficiency of NHEJ. Following the initial crisis, parasites that underwent modification by CRISPR/Cas9 showed no secondary phenotypes in IDE 2 growth rate, plaque morphology, or replication that would suggest off-target effects of the CRISPR/Cas9 system. However, as with the severe bottlenecks that occur during antibiotic selection, genetic complementation will need to be performed to account for the presence of secondary mutations or polar effects. The vulnerability of DKU80 strains to targeted double-strand breaks suggested we could perhaps rescue them through homologous recombination and, in the process, introduce desired point mutations. We observed that a 90 bp oligonucleotide homologous to the targeted region of PKG indeed improved the survival rate of DKU80 parasites transfected with pU6-PKG. Furthermore, a mutation included in the oligonucleotide was successfully incorporated into the parasite genome in a third of the surviving population. Similarly, targeting of the 39 end of the CDPK3 locus allowed us to incorporate an epitope tag into the open reading frame, which could be observed in 15% of the population three days post transfection, again in the absence of selection. As predicted the rate of endogenous tagging was significantly reduced in wild-type parasites, consistent with the NHEJ-mediated repair of targeted double-stranded breaks in this strain.