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

Bio-protocol
Azat AkhmetovEdward M Marcotte

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.

References

Apr 5, 2002·Genome Research·W James Kent
May 29, 2009·DNA Repair·Eleni P Mimitou, Lorraine S Symington
Aug 16, 2011·Nature Structural & Molecular Biology·Xuefeng ChenGrzegorz Ira
Mar 6, 2013·Nucleic Acids Research·James E DiCarloGeorge M Church
Apr 15, 2015·ACS Synthetic Biology·Michael E LeeJohn E Dueber

❮ Previous
Next ❯

Citations

Nov 7, 2020·Frontiers in Bioengineering and Biotechnology·Eugene Fletcher, Kristin Baetz

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
PCRs
electrophoresis

Software Mentioned

CRISP
Geneious
MoClo Toolkit
BLAT
Mo Clo

Related Concepts

Related Feeds

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.