title: New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.
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.
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: Marian F
authors: MF
authors: Laughery
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: Tierra
authors: T
authors: Hunter
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: Alexander
authors: A
authors: Brown
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: James
authors: J
authors: Hoopes
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: Travis
authors: T
authors: Ostbye
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
authors: Taven
authors: T
authors: Shumaker
authors: School of Molecular Biosciences, Washington State University, Pullman, WA, USA.|Center for Reproductive Biology, Washington State University, Pullman, WA, USA.
authors: John J
authors: JJ
authors: Wyrick
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019295:Computational Biology / Q000379:methods; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D017393:RNA Editing / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D064130:CRISPR-Associated Proteins; D005819:Genetic Markers; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D004720:Endonucleases
keywords: CRISPR; Cas9; Saccharomyces cerevisiae; genome editing; guide RNA; plasmids