Rapid CRISPR–Cas9 Genome Editing in S. cerevisiae

bioRxiv (Cold Spring Harbor Laboratory) 2026 Preprint Open access

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Authors: Hosein Rostamian, Ethan W. Madden, Frank M. Kaplan, Riley Kim, Daniel G. Isom, Brian D. Strahl
DOI: 10.64898/2026.03.27.714888
Cited by: 1
References: 9

Abstract

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.

Fields of Study & Subjects

CRISPR and Genetic Engineering Genetic Neurodegenerative Diseases Microbial Metabolic Engineering and Bioproduction

Publication details

Journal / source
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Publication date
30 March 2026
Language
EN
Document type
Preprint