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Published on: May 28, 2019
Rapid CRISPR-Cas9 Genome Editing in S. cerevisiae
Hosein Rostamian1,2, Ethan W Madden1,2, Frank M Kaplan1
1Department of Biochemistry & Biophysics, The University of North Carolina School of Medicine, Chapel Hill, NC, 27599, USA.
None:
This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.
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