Although we cannot be certain that the different instances represent independent events, repeated 6 bp sequences flanking the deletion suggest that the same rearrangement could have occurred various times through homologous recombination. We speculated that one potential cause for the observed decrease in gene CBiPES hydrochloride disruption following OR-486 transfection of DKU80 parasites could be an inability to repair double stranded breaks. Consistent with this idea, these strains display increased sensitivity to DNA-damaging agents. To test this hypothesis, we performed plaque assays to measure parasite viability immediately after transfection. We compared the number of plaques formed by mock-transfected parasites to those formed following transfection with pU6-SAG1 or either of two control plasmids. Both control plasmids were based on the pU6-SAG1 construct. The first plasmid lacks a protospacer specific to the T. gondii genome, and the Cas9 nuclease has been replaced with a pyrimethamine resistance cassette. The second plasmid only differs from the pU6- SAG1 plasmid in that it lacks the protospacer. All DNA transfections decreased the viability of parasites, although this effect was smallest in wild-type parasites transfected with either of the control plasmids. The DKU80 strain was more susceptible than wild type to transfection with either the control plasmids or pU6-SAG1. Expression of the nuclease without a targeting protospacer had a marginally increased cost to viability that only achieved significance in the DKU80 strain. In both strains, the greatest loss of viability was observed upon transfection with pU6-SAG1, which, as opposed to the controls, is expected to efficiently introduce double-stranded breaks at the SAG1 locus. This suggests that loss of viability in response to CRISPR/Cas9 is multifactorial, including a modest effect from nuclease expression that is significantly exacerbated by the presence of a protospacer targeting the genome. At present, we cannot explain the decrease in DKU80 viability upon transfection with the control plasmids. However, consistent with its increased susceptibility to DNA damage, the DKU80 strain may be unable to recover from CRISPR/Cas9-targeted double-stranded breaks in the absence of a template for homologous recombination, effectively resulting in a reduced frequency of observed gene disruption. Current methods for genome engineering of T. gondii rely on the generation of complex constructs for homologous recombination, which yield significant efficiency only in specific parasite strains and require antibiotic selection.