Seamless site-directed mutagenesis of the Saccharomyces cerevisiae genome using CRISPR-Cas9

Abstract

CRISPR assisted homology directed repair enables the introduction of virtually any modification to the Saccharomyces cerevisiae genome. Of obvious interest is the marker-free and seamless introduction of point mutations. To fulfill this promise, a strategy that effects single nucleotide changes while preventing repeated recognition and cutting by the gRNA/Cas9 complex is needed. We demonstrate a two-step method to introduce point mutations at 17 positions in the S. cerevisiae genome. We show the general applicability of the method, enabling the seamless introduction of single nucleotide changes at any location, including essential genes and non-coding regions. We also show a quantifiable phenotype for a point mutation introduced in gene GSH1. The ease and wide applicability of this general method, combined with the demonstration of its feasibility will enable genome editing at an unprecedented level of detail in yeast and other organisms.

Keywords: CRISPR-Cas9; Genome editing; Saccharomyces cerevisiae; Site-directed mutagenesis.

Fig. 1

Outline of the two-step, stuffer-assisted genome site-directed mutagenesis strategy. Two variations of the strategy were applied. In the stuffer strategy a protospacer target sequence located near the site to mutagenize is replaced by a heterologous 20-nucleotide sequence (the stuffer) by CRISPR-Cas9 assisted homologous recombination, leaving the PAM site intact. The stuffer may be a standard, randomly generated sequence (the standard stuffer, left box) or a degenerate sequence bearing at least seven mismatches with the original protospacer (a silent stuffer, middle box). The second CRISPR step uses the stuffer as a protospacer, restoring the original protospacer sequence and introducing the desired mutation in a single homologous recombination event. A second variation on the strategy replaces the entire target ORF – or nearby ORF if an intergenic region is the target of mutagenesis – by a heterologous stuffer ORF (e.g. GFP), which is targeted by one or more gRNAs in the second step (right box). Homologous recombination restores the original ORF with mutations. Successful integration is easily assessed by PCR. The strategies were tested at the positions indicated in the boxes. Positions are identified by the coordinate of the first nucleotide of the PAM site (NGG) with respect to the nearest ORF. For each position, stuffer insertion was successful in either all strains tested (green check), two out of three strains (yellow check), in all strains but led to a slow growth phenotype (yellow-x), or in none of the strains (red-x). Once a stuffer was inserted, its removal and replacement by the point mutant sequence was successful in all cases

Fig. 2

Point mutations introduced with the stuffer-assisted genome site-directed mutagenesis method lead to detectable phenotypic changes. a Simplified representation of glutathione synthesis and recycling. Condensation of glutamate and cysteine by Gsh1p is followed by the addition of a glycine by Gsh2p, yielding reduced glutathione. Glutathione oxidized by reactive oxygen species (ROS) is recycled to its reduced form by the NADPH-dependent Glr1p enzyme. b Sequencing shows successful insertion of the stuffer and subsequent introduction of a point mutation in the GSH1 promoter sequence c ROS accumulation induced by exposure to SSL was compared between a gsh1(A(−73)T) point mutant generated with the method, and in its parent wildtype strain (WT). ROS accumulation was assessed using flow cytometry, measuring the mean fluorescence of cells treated with CellROX Deep Red reagent. ROS were measured 16 h after inoculation in minimal medium (Mid-log), after overnight incubation in undiluted SSL (acute stress), or after 24 and 48 h in minimal medium containing 70 % SSL