CRISPR/Cas9‐Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction‐Free Cloning and a Rapid BamHI Digest Readout. Issue 21 (9th December 2020)

• Record Type: Journal Article
• Authors: Sirois, Allison R.; Pilotte, Nils; Williams, Steven A.; Saunders, Lori J.
• Is Part Of: Current protocols essential laboratory techniques. Issue 21(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-12-09
• Subjects: ADE2 -- Cas9 -- CRISPR -- gene editing -- restriction‐free cloning -- yeast; Life sciences -- Laboratory manuals; Medical laboratories; Diagnosis, Laboratory; Clinical Laboratory Techniques; Clinical Protocols; Diagnosis, Laboratory; Medical laboratories; 570.28
• DOI: 10.1002/cpet.45
• Languages: English
• ISSNs: 1948-3430

Abstract

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated systems (CRISPR/Cas) are revolutionary tools for predictable and repeatable genome editing. In this article, the CRISPR/Cas9 system will be used to engineer the genome of the yeast Saccharomyces cerevisiae . As a model organism utilized across biological disciplines, efficient and tailorable methods for engineering the yeast genome are indispensable for a variety of research, educational, and commercial applications. The protocols described here incorporate a simple restriction‐free cloning strategy and digestion‐based screening method that can be readily and easily modified to suit user‐designed experimental needs. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Recombinant pCAS plasmid preparation Basic Protocol 2 : Barcode/editing fragment assembly Basic Protocol 3 : Gene editing by yeast co‐transformation Alternate Protocol : Competent yeast preparation and transformation by a lithium acetate/single‐stranded carrier method