CRISPR-Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains

Abstract

There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR-Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.

Keywords: Biorefineries; CRISPR–Cas9; CRISPR–Cas9, clustered regularly interspaced short palindromic repeats–CRISPR-associated endonuclease 9; Chemical production; DSB, double strand break; GOI, gene of interest; Genome editing; HDR, homology-directed repair; HR, homologous recombination; Industrial yeast; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; PI, propidium iodide; SNPs, single nucleotide polymorphisms; TALENs, transcription activator-like effector nucleases; USER, uracil-specific excision reaction; ZFNs, zinc finger nucleases; crRNA, CRISPR RNA; gRNA, guide RNA; tracrRNA, trans-activating RNA.

Fig. 1

Determination of strain ploidy. Uniparametric histogram shows distribution of PI stained cells according to their relative DNA content as examined by flow cytometry. CEN.PK113-7D strain of known ploidy was included as a reference. Individual histograms overlaid with 1N (haploid) CEN.PK histogram for comparison are included at the upper part of the picture. First peak represents population of G1-phase cells, second peak population of G2-phase cells.

Fig. 2

Tools for Cas9-mediated genome editing in industrial strains. (A) Schematic illustration of replicative (CEN/ARS containing) plasmid carrying Cas9 gene, controlled by TEF1 (or ADH1) promoter, and kanMX marker for selection. Alternatively, insertion of P_TEF1-Cas9 into the genome using integrative plasmid (not displayed) was performed. The vector with TEF1 promoter controlling Cas9 expression was used unless otherwise stated. The two-micron-based replicative plasmid contains the gRNA expression cassette and natMX dominant marker for selection. The detailed illustration of gRNA cassette is displayed below the plasmid maps, including design of phosphorylated (P) primers used for PCR amplification of the plasmid with forward primer containing specific 20-bp target sequence (TS). (B) Illustration of target site of a gene of interest (GOI) in the genome, Cas9 target site being part of gRNA sequence and followed by PAM motif is highlighted. Templates, 90-bp dsDNA oligo introducing STOP codon into the coding sequence (1) and a gene expression cassette disrupting the coding sequence (2), used for DSB repair are displayed below.

Fig. 3

Disruption of ADE2 gene – proof-of concept. (A) Illustration of Cas9-mediated genomic changes in ADE2 coding sequence. Introduction of STOP codon into coding sequence of ADE2 gene in haploid – 1n and diploid – 2n cells, left; disruption of ADE2 sequence with P_TEF1-GFP expression cassette, right. (B) Colonies of the strains with single-allele ADE2 disruption performed with PCR-generated marker cassette disruption. Red color of the colony represents ade2Δ phenotype. (C) Colonies of the Cas9-expressing strains selected for presence of gRNA plasmid, being transformed along with 1 nmol/μl 90-bp dsDNA donor. (D) Disruption frequency of ADE2 gene (number of red colonies per total number of transformants selected for presence of both Cas9-carrying and gRNA-carrying vectors) in analyzed strains. Black columns show the mutation frequency when dsDNA oligo donor is used as a repair template and Cas9 expression is driven by TEF1 promoter, dashed columns represent the situation when Cas9 expression is driven by ADH1 promoter, gray columns show the mutation frequency when 15 μg of P_TEF1-GFP cassette is used instead of dsDNA oligo donor and white columns represent the case when any repair template is omitted. Green pattern columns show percentage of fluorescent clones among ade2Δ mutants. Error bars represent standard deviation (SD, N=3 or N=2 in case of ADH1p-Cas9 experiment).

Fig. 4

Construction of lactic acid producing yeast strains. (A) Illustration of one-step Cas9-mediated PDC1 and PDC5 gene disruption in strains containing ldhL gene inserted in chromosome X, left; and PDC1 and PDC5 disruption by P_TEF1-ldhL expression cassette, right. Haploid strain – 1n, diploid strain – 2n. (B) Time-course metabolite profile (lactic acid – closed triangles, glucose – closed diamonds, pyruvate – dashed line with open triangles, ethanol – closed squares, acetate – closed circles) and growth properties (OD – dashed line with closed diamonds) of strains engineered for production of lactic acid. Glucose concentration is plotted on the secondary y axis. Upper charts represent CEN.PK derived strains. Lower charts represent Ethanol Red derived strains. Genotypes of the strains are in the left corner of each chart, P_TEF1-ldhL pdc1Δ pdc5Δ represents strains containing ldhL gene inserted in chromosome X and non-sense mutations in PDC1 and PDC5 genes, P_TEF1-ldhL: : pdc1Δ P_TEF1-ldhL: : pdc5Δ represents strains with disruption of PDC1 and PDC5 by P_TEF1-ldhL expression cassette. Samples from three biological replicates were taken at marked time points. Error bars represent standard deviation (SD, N=3).