1 Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
2 Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
3 Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University Langone Health, New York, NY, USA.
4 The Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montreal, QC, Canada.

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

Bio Protoc. 2018 Mar 20;8(6):

Authors: Akhmetov A, Laurent JM, Gollihar J, Gardner EC, Garge RK, Ellington AD, Kachroo AH, Marcotte EM

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.

PMID: 29770349 [PubMed]