CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

TLDR

A variety of strain‑engineering techniques have been developed for Saccharomyces cerevisiae, yet constructing strains with multiple genetic changes remains time‑consuming. The authors aim to create CRISPR/Cas9‑based methods that enable rapid, simultaneous introduction of multiple genetic modifications in yeast. They present an open‑source web tool for identifying Cas9 target sites and two transformation strategies—an in‑vivo assembly of a guide‑RNA plasmid and an in‑vitro assembly of plasmids carrying two gRNAs—to achieve high‑accuracy, multi‑modification editing. The approaches achieved high‑accuracy editing, enabling simultaneous integration of a multigene construct with gene deletion and two single‑nucleotide mutations, and allowed up to six genetic modifications in a single transformation, with standardized plasmids and tools released for community use.

Abstract

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.