A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast

CRISPR/Cas9 technology allows accurate, marker-less genome editing. We report a detailed, robust, and streamlined protocol for CRISPR/Cas9 genome editing in Saccharomyces cerevisiae, based on the widely used MoClo-Yeast Toolkit (https://www.addgene.org/kits/moclo-ytk/). This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group. For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021). [Display omitted] •Complete and user-friendly protocol for CRISPR/Cas9 yeast genome editing•sgRNA(s) and Cas9 cloning via Golden Gate Assembly with the MoClo-Yeast Toolkit•Detailed description of repair fragment design for several genome editing applications•Yeast one-step co-transformation with sgRNA(s)+Cas9 plasmid and repair fragment Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. CRISPR/Cas9 technology allows accurate, marker-less genome editing. We report a detailed, robust, and streamlined protocol for CRISPR/Cas9 genome editing in Saccharomyces cerevisiae, based on the widely used MoClo-Yeast Toolkit (https://www.addgene.org/kits/moclo-ytk/). This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group.