The yeast S. cerevisiae is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the performing of a wide variety of genome modifications. Among the latter, one can mention point mutations, disruptions and deletions of particular genes and regions of chromosomes, insertion of cassettes for the expression of heterologous genes, targeted chromosomal rearrangements such as translocations and inversions, directed changes in the karyotype (loss or duplication of particular chromosomes, changes in the level of ploidy), mating-type changes, etc. Classical yeast genome manipulations have been advanced with CRISPR/Cas9 technology in recent years that allow for the generation of multiple simultaneous changes in the yeast genome. In this review we discuss practical applications of both the classical yeast genome modification methods as well as CRISPR/Cas9 technology. In addition, we review methods for ploidy changes, including aneuploid generation, methods for mating type switching and directed DSB. Combined with a description of useful selective markers and transformation techniques, this work represents a nearly complete guide to yeast genome modification.

The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases. New approaches to alleviate the risks associated with genome editing include attenuating the off-target activity of editing complex through the use of modified forms of Cas9 nuclease and single guide RNA (sgRNA), improving delivery methods for sgRNA/Cas9 complex, and directing DNA lesions caused by the sgRNA/Cas9 to non-mutagenic repair pathways. Here, we have described CRISPR/Cas9 as a new powerful mutagenic factor, discussed its mutagenic properties, and reviewed factors influencing the mutagenic activity of CRISPR/Cas9.

CRISPR/Cas9-based genome editing systems are widely used for genetic modification of different organisms. One of the drawbacks of CRISPR/Cas9 methods is the non-specific activity of Cas9, which can lead to accumulation of unwanted mutations in the edited genome [1]. Therefore, the development ofin vivomodels for high-throughput analysis of factors influencing the frequency of mutagenesis associated with the use of CRISPR/Cas9 technologies is a relevant task. YeastSaccharomycescerevisiaeis a convenient object for developing such models [2]. Here we represent a yeastin vivomodel that allows us to evaluate the effects of nucleotide sequence of the protospacer adjacent motif (PAM) and the guide RNA (gRNA) on the efficiency of binding between the gRNA/Cas9 complex and the target sequence in the genome. Since the Cas9 activity is lethal in cells lacking a donor sequence for homologous repair of double-strand breaks caused by this endonuclease, in the proposed test-system, the reduced efficiency of transformation by a plasmid encoding Cas9 and various gRNA variants reflects the efficiency of recognition of the target gene by the gRNA/Cas9 complex. To study the influence of different PAM variants, with a consensus of NGG, on CRISPR/Cas9 activity, we obtained four isogenic strains that differ in their PAM sequence (AGG, TGG, CGG, GGG) in the codon 202 of the chromosomal copy of the reporter geneURA3. To evaluate the effect of incomplete matching between gRNA and the target site sequences, we propose using a series of plasmids based on the pML107 vector, encoding Cas9 and one of the 20 possible gRNA variants with single nucleotide substitutions at each of the 20 positions. The results obtained so far indicate the potential of the proposed approach.

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.