A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9.

TitleA Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9.
Publication TypeJournal Article
Year of Publication2022
AuthorsAguilar RR, Shen Z-J, Tyler JK
JournalMethods Protoc
Date Published2022 Oct 04

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.

Alternate JournalMethods Protoc
PubMed ID36287051
PubMed Central IDPMC9607540
Grant ListR01 AG079883 / AG / NIA NIH HHS / United States
R35 GM139816 / GM / NIGMS NIH HHS / United States
Related Faculty: 
Jessica K. Tyler, Ph.D.

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