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Updated: Aug 5, 2026
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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
Published on: May 28, 2019
Protocol for CRISPR genome editing in S. cerevisiae using PCR-based guide insertion
Hosein Rostamian1, Ethan W Madden1, Frank M Kaplan2
1Department of Biochemistry & Biophysics, The University of North Carolina School of Medicine, Chapel Hill, NC, USA; Curriculum in Genetics and Molecular Biology, The University of North Carolina School of Medicine, Chapel Hill, NC, USA.
STAR Protocols
|July 28, 2026
Summary
This study introduces a faster CRISPR-Cas9 genome editing method for yeast. It simplifies guide RNA construction and speeds up experiments by using PCR instead of traditional cloning techniques.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 technology enables precise genome editing.
- Traditional methods for CRISPR-Cas9 in Saccharomyces cerevisiae can be time-consuming.
- Efficient genome engineering is crucial for yeast research and biotechnology.
Purpose of the Study:
- To develop an accelerated CRISPR-Cas9 genome editing protocol for Saccharomyces cerevisiae.
- To streamline the process of guide RNA construction and installation.
- To facilitate rapid iteration of genetic modifications in yeast.
Main Methods:
- Replaced restriction/ligation cloning with PCR-based guide installation.
- Developed seamless Cas9-plasmid recircularization.
- Utilized a KanMX/G418-selectable Cas9-sgRNA plasmid.
- Employed lithium acetate/polyethylene glycol (LiAc/PEG) co-transformation for yeast.
Main Results:
- Successfully implemented a simplified and rapid CRISPR-Cas9 genome editing protocol.
- PCR-based installation and seamless recircularization accelerated guide construction.
- Verified plasmid cloning and sequence in E. coli prior to yeast transformation.
- Demonstrated efficient co-transformation of yeast with the Cas9-sgRNA plasmid and HDR donor.
Conclusions:
- The presented protocol significantly simplifies and accelerates CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.
- This method allows for rapid guide iteration, enhancing experimental efficiency.
- The optimized protocol is valuable for genetic studies and synthetic biology applications in yeast.
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