CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression.IMPORTANCEToxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.