Cas9 Endonuclease Toxicity in Haploid and Diploid Strains of Saccharomyces cerevisiae

Objective: CRISPR/Cas9-based RNA-guided genome-editing tools are highly effective. However, off-target mutations and cellular toxicity limit their utility. The main goal of the study is to investigate whether Cas9 exhibits toxicity towards budding yeast Saccharomyces cerevisiae. Methods: Haploid and diploid S. cerevisiae strains were engineered to express either Cas9 or both Cas9 and a single-guide RNA (sgRNA). Growth curve analysis was used to evaluate the impact of Cas9 expression on yeast viability. Results and Discussion: Haploid and diploid strains expressing both Cas9 and sgRNA exhibit lower growth rates compared to strains that do not produce either Cas9 or sgRNA, presumably due to effective double-strand breaks induction. Toxic effect of Cas9 without sgRNA was observed as well. Cas9 toxicity appears more severe in haploid yeast while diploid strain is more tolerant to Cas9 overproduction. Toxicity of Cas9 may be caused by its non-specific binding to genomic DNA or excessive protein production. Conclusions: The Cas9 endonuclease is toxic in S. cerevisiae. This may compromice the efficacy of the CRISPR/Cas9 editing system. Therefore, mitigating Cas9 toxicity should be one of the strategies for improving genome editing outcomes.