New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing.

Keywords: CRISPR; Cas9; Saccharomyces cerevisiae; genome editing; guide RNA; plasmids.

Figure 1

Design of new guide RNA expression cassette for rapid cloning of 20mer guide sequences. Unique BclI and SwaI sites enable efficient cloning of any 20mer guide RNA targeting sequence into the single guide RNA (sgRNA) expression cassette. A plasmid with the guide RNA expression cassette is linearized by digestion with BclI and SwaI enzymes. Oligonucleotides are designed to contain a compatible GATC overhang, a 20mer guide sequence (in this case targeting the yeast TRP1 gene), and the 5' end of the structural segment of the sgRNA. The hybridized oligonucleotides are ligated into the digested plasmid, yielding the final complete sgRNA expression cassette. The asterisk indicates that BclI cutting is blocked by Dam methylation.

Figure 2

Maps of new plasmids for CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. (A and B) Vectors for two-plasmid system of CRISPR-Cas9 gene editing. pT040 has the guide RNA expression cassette with unique BclI-SwaI restriction enzyme sites for cloning the 20mer guide sequence. pJH001 is a high-copy Cas9 expression vector. (C and D) URA3 and LEU2 vectors for one-plasmid system of CRISPR-Cas9 gene editing. Each vector contains the Cas9 gene and the guide RNA expression cassette with unique BclI-SwaI restriction enzyme sites for cloning the 20mer guide sequence (see Figure 1). The asterisk indicates that BclI cutting is blocked by Dam methylation.

Figure 3

Targeting the yeast TRP1 gene for CRISPR-Cas9 gene editing. The genomic DNA (gDNA) region targeted by the guide sequence is indicated, along with the location of the Cas9 cut site. The TRP1 start codon is underlined. A portion of the 90mer oligonucleotide template with the targeted TRP1 mutation is shown. Recombination between the gDNA and template eliminates the PAM sequence adjacent to the Cas9 cut site and introduces an early frame shift in the TRP1 gene.