Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

Figure 1.

Diagram of Cas9 complex and schematic of genetic constructs. (A) Illustration of Cas9 protein interacting with CRISPR gRNA to direct endonuclease activity proximal to the PAM sequence. (B) Design of the Cas9 and gRNA constructs. Cas9 gene contained a SV40 nuclear localization signal and was expressed under the Gal-L inducible promoter in CAN1 experiments and the TEF1 constitutive promoter in ADE2 experiments. The gRNA was expressed under the snoRNA SNR52 promoter and contained a terminator from the 3′ region of the yeast SUP4 gene. CAN1.Y and CAN1.Z were targeted to different loci in the CAN1 gene, whereas ADE2.Y and ADE2.Z were targeted to different loci in the ADE2 gene.

Figure 2.

CRISPR-Cas mediated genomic mutagenesis. (A) Schematic of the CAN1.Y and CAN1.Z genomic target sequences and respective PAM sequences in the CAN1 gene. (B) Percentage survival of yeast strains expressing different combinations of Cas9 and gRNA. Only combination of Cas9 and gRNA caused decrease in cell viability. Error bars represent standard deviation between four experiments. (C) CAN1 and LYP1 mutation frequency of yeast strains expressing different combinations of Cas9 and gRNA on galactose induction of Cas9. Mutation frequency of CAN1 (the gRNA-targeted gene) is elevated in strains expressing Cas9 and gRNA, whereas LYP1 (not targeted by gRNA) remained constant. Error bars represent standard deviation between four experiments. (D) Alignments of CAN1 gene from canavanine-resistant colonies post-galactose induction of Cas9 in gRNA-expressing strains. The underlined sequence is of the wild-type reference CAN1 gene, whereas the following eight sequences are from colonies from the population. The PAM sequence is highlighted in purple, whereas the gRNA guiding sequence is highlighted in blue. Nonsense mutations observed are largely deletions or insertions 5′ to the PAM sequence.

Figure 3.

The gRNA and donor DNA recombination in Cas9 constitutively expressing strains. (A) Diagram of ADE2 gene in the 5′–3′ direction displaying the gRNA genomic target regions and their respective PAM sequences. The sequence location of the single- or double-stranded oligonucleotide that will repair the premature stop codon is also displayed. The single-stranded oligonucleotide is from the non-coding strand of the ADE2 gene, whereas the double-stranded oligonucleotide is composed of annealed non-coding and coding strands. The oligonucleotide was designed centred around the mutant base pair. The gRNA sequences were designed based on the closest PAM sequences to the premature stop codon. (B) 90 mer single- and double-stranded oligonucleotides were electroporated into yeast expressing Cas9 under the TEF1 constitutive promoter, with and without a gRNA cassette. The gRNA cassette is a PCR product consisting of the SNR52 promoter, the genomic target, the guiding RNA scaffold and the SUP4 3′ flanking sequence. Electroporation of oligonucleotide and ADE.Y gRNA cassette increases oligonucleotide incorporation up to 5-fold for single-stranded oligonucleotide and 130-fold for double-stranded oligonucleotide. Approximately 10⁶–10⁷ cells were plated on selective media to monitor ADE2 correction frequency. Values are averages from four experiments, and error bars represent standard deviation from the experiments. (C) Transformation frequency of plasmid with and without gRNA CAN1.Y expression in cells containing Cas9 constitutively expressed on a second plasmid. Transformation frequency is defined by the ratio of number colonies that recover on defined media to select for both plasmids divided by the number of colonies on rich non-selective media. The plasmid transformation frequency decreases when gRNA expression is present on the plasmid, as expected owing to cell death associated with Cas9 DNA cleavage. (D) Percentage of cells containing gRNA and constitutive Cas9 plasmids resistant to Canavanine and G418 via replica plating. When a CAN1.Y double-stranded oligonucleotide or a KanMX resistance cassette targeted for disruption of the CAN1 and the CAN1.Y PAM sequence were co-transformed with the gRNA expression plasmid, nearly 100% of the cells selected for Cas9 and gRNA plasmids were resistant to canavanine. Furthermore, in the case of the KanMX resistance cassette donor DNA, the same percentage of cells resistant to canavanine was also resistant to G418, indicating all KanMX insertions integrated in the targeted locus. Values for Figure 3C and D represent an average of three experiments for the no donor DNA controls and six experiments for the transformations containing donor DNA. Error bars represent standard deviation of values.