A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.

Nicotinamide Riboside and Metformin Ameliorate Mitophagy Defect in Induced Pluripotent Stem Cell-Derived Astrocytes With POLG Mutations

“…We are fully aware that the current state of the art for iPSC studies is to use gene-corrected isogenic controls; however, the presence of compound mutations, such as those used in this study (CP2A mutation), makes the generation of individual controls impracticable. At the same time, it has been stated that the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by the standard CRISPR/Cas9 technique near impossible (EauClaire and Webb, 2019;Liang et al, 2020aLiang et al, ,b, 2021.…”

Stress tolerance enhancement via SPT15 base editing in Saccharomyces cerevisiae

“…These results indicated that gene- and site-specific biases were also observed for Target-AID-mediated point mutagenesis. However, compared to site-specific point mutagenesis by CRISPR/Cas9 practices [ 54 – 56 ], the Target-AID system avoids high cell toxicity and some tedious processes, such as design and usage of heterology block or stuffer to prevent repeated recognition and cutting by the gRNA/Cas9 complex, preparation of donor DNAs, etc. Therefore, Target-AID could provide a powerful and effective tool to generate site-specific point mutagenesis in S. cerevisiae .…”

Stress tolerance enhancement via SPT15 base editing in Saccharomyces cerevisiae

“…These results indicated that gene-and site-speci c biases were also observed for Target-AID-mediated point mutagenesis. However, compared to sitespeci c point mutagenesis by CRISPR/Cas9 practices [54][55][56], the Target-AID system avoids high cell toxicity and some tedious processes, such as design and usage of heterology block or stuffer to prevent repeated recognition and cutting by the gRNA/Cas9 complex, preparation of donor DNAs, etc. Therefore, Target-AID could provide a powerful and effective tool to generate site-speci c point mutagenesis in S. cerevisiae.…”