Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9

Abstract

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.

Systematic humanization of the yeast cytoskeleton discerns functionally replaceable from divergent human genes

“…S2A ) (ii) inactivated via a temperature-sensitive allele 22, 36 ( Fig. S3A ) or (iii) endogenously replaced by homologous recombination repair with the human ortholog(s) following CRISPR-Cas9 cleavage 8, 37 ( Fig. S4A ).…”

“…S3B, C ). Finally, in cases of endogenously chromosomal replacement, human genes were assayed by natively substituting their yeast ORF mediated via CRISPR-Cas9 37 ( Fig. S4B, C ).…”

“…29 human genes were assayed using a CRISPR-Cas9 mediated yeast genome editing protocol as previously described 8, 37 . For every yeast gene to be replaced endogenously, a minimum of 2 synthetic guide RNAs (sgRNA) with high ON-target and low OFF-target scores were designed using the Geneious v10.2.6 CRISPR-Cas9 tools suite.…”

“…Transformants were selected on SC-URA medium to select for the knockout plasmid. While CRISPR-mediated double-stranded breaks are intrinsically lethal, targeted editing of an essential gene locus acts as an added layer of selection in replacing the human gene of interest, allowing cells to survive only if the human ortholog being assayed functionally replaces the yeast gene at the appropriate locus 8, 37 . Surviving colonies ( Fig.…”

Precise replacement ofSaccharomyces cerevisiaeproteasome genes with human orthologs by an integrative targeting method

“…Finally, when using CRISPR-Cas9, a specific repair event can be selected from all possible events only if it confers a novel selectable phenotype. When the DSB is induced within an essential gene, the selection for repair to a viable state is strong 29 , but changes made at nonessential loci lack that advantage.…”