A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae.

Peter W Daniels,Bin Hu,Anuradha Mukherjee,Alastair Sh Goldman

doi:10.12688/wellcomeopenres.14642.2

Abstract

Integrating a desired DNA sequence into yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans.

Highlights

The budding yeast Saccharomyces cerevisiae has been widely used as a model organism for basic research in all aspects of eukaryotic biology
The principle of this system is that the desired DNA sequence is integrated into the yeast genome as a consequence of homologous repair of a double-strand break (DSB) generated at a specific site by the CRISPR-Cas[9] system
We constructed two pRS425-based episomal plasmids, pBH263 and pBH750, each bearing expression cassettes for Cas[9] and Lys2/Met15-targeting guide RNA (gRNA) (Figure 2A). When these plasmids were introduced into yeast cells, a DSB was produced inside the Lys[2] or Met[15] gene by Cas[9]

Summary

Introduction

The budding yeast Saccharomyces cerevisiae has been widely used as a model organism for basic research in all aspects of eukaryotic biology. The most commonly used nutritional markers are the URA3, LEU2, TRP1, and HIS3 genes, which are required for synthesis of pyrimidine, L-leucine, L-tryptophan, and L-histidine respectively To integrate these vectors into yeast genome, these circular plasmids are first linearized by restriction digestion occurring inside the nutritional marker genes. It is important that the host yeast cells should harbor the corresponding auxotrophic mutation or deletion ( called auxotrophic marker) for the selection of the successful transformants. The consequence of this integration is the target DNA sequence being flanked by a mutated allele and a wild-type allele of the nutritional marker. The yeast cells acquiring the integrative vector become prototrophic for the corresponding nutritional marker[4]

Methods

Results

Conclusion