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

Aug 26, 2015Yeast (Chichester, England)

New tools for easier CRISPR gene editing in baker’s yeast

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Abstract

A new sgRNA expression cassette facilitates rapid cloning of 20mer guide sequences for genome editing in yeast.

CRISPR-Cas9 technology allows targeted DNA double-strand breaks, important for genome editing.
Previous methods for introducing guide sequences into yeast often involve complex and costly cloning procedures.
The new expression cassette includes internal restriction enzyme sites for easier and faster cloning.
A specific vector with the URA3 selectable marker (pML104) may enhance genome editing efficiency and flexibility in yeast.
The simplified vectors could accelerate the adoption of CRISPR-Cas9 technology in yeast genome editing.

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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.

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

Abstract

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