Journal article
New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae
Yeast (Chichester, England), Vol.32(12), pp.711-720
12/2015
Handle:
https://hdl.handle.net/2376/102891
PMCID: PMC4715497
PMID: 26305040
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.
Metrics
13 Record Views
Details
- Title
- New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae
- Creators
- Marian F Laughery - School of Molecular Biosciences, Washington State University, Pullman, WA, USATierra Hunter - School of Molecular Biosciences, Washington State University, Pullman, WA, USAAlexander Brown - School of Molecular Biosciences, Washington State University, Pullman, WA, USAJames Hoopes - School of Molecular Biosciences, Washington State University, Pullman, WA, USATravis Ostbye - School of Molecular Biosciences, Washington State University, Pullman, WA, USATaven Shumaker - School of Molecular Biosciences, Washington State University, Pullman, WA, USAJohn J Wyrick - Center for Reproductive Biology, Washington State University, Pullman, WA, USA
- Publication Details
- Yeast (Chichester, England), Vol.32(12), pp.711-720
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- England
- Grant note
- R37 ES002614 / NIEHS NIH HHS R01 ES002614 / NIEHS NIH HHS ES002614 / NIEHS NIH HHS
- Identifiers
- 99900546678101842
- Language
- English
- Resource Type
- Journal article