Optimizing Systems for Cas9 Expression in Toxoplasma gondii.

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• CRISPR-Cas9 technology enables genome engineering but can cause toxicity, limiting its application in certain organisms.
• Cas9 toxicity has been observed in various species, including the apicomplexan parasite *Toxoplasma gondii*, affecting experimental outcomes.
• Previous work identified a single guide RNA (sgRNA) that partially mitigated Cas9 toxicity in *T. gondii*, facilitating genome-wide screens.

Purpose of the Study:

• To define the requirements for stable Cas9 expression by comparing different expression constructs.
• To characterize the role of the buffering sgRNA in understanding and alleviating Cas9 toxicity.
• To develop guidelines for improved Cas9 expression stability and selection stringency for diverse genetic applications.

Main Methods:

• Comparison of various Cas9 expression constructs, including those utilizing viral 2A peptides.
• Characterization of the dual functions of the buffering sgRNA in genome targeting and parasite fitness.
• Assessment of Cas9 expression stability and selection stringency across different experimental conditions.

Main Results:

• Viral 2A peptides significantly enhance the selection and stability of Cas9 expression.
• The buffering sgRNA plays a dual role: facilitating initial construct integration and improving long-term parasite fitness by reducing Cas9 toxicity.
• Guidelines for stable Cas9 expression have been established, applicable to various genetic approaches in diverse organisms.

Conclusions:

• Viral 2A peptides are effective in improving Cas9 expression stability and selection.
• The sgRNA is crucial for both initial genome targeting and mitigating Cas9 toxicity, enhancing experimental reliability.
• This research provides a framework for optimizing CRISPR-Cas9 applications and highlights the importance of characterizing Cas9 expression effects.