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

Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21.

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.

Keywords: CRISPR; Cas9; Saccharomyces cerevisiae; genome editing; guide RNA; plasmids.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Computational Biology / methods
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • Endonucleases / genetics
  • Gene Expression
  • Gene Targeting
  • Genetic Markers / genetics
  • Genetic Vectors*
  • Polymerase Chain Reaction
  • RNA Editing / genetics*
  • RNA, Guide / genetics
  • Saccharomyces cerevisiae / genetics*
  • Transformation, Genetic

Substances

  • CRISPR-Associated Proteins
  • Genetic Markers
  • RNA, Guide
  • DNA
  • Endonucleases