CRISPR/Cas9‐Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction‐Free Cloning and a Rapid Bam HI Digest Readout

CRISPR/Cas9‐Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction‐Free Cloning and a Rapid Bam HI Digest Readout

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...

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Veröffentlicht in:Current Protocols Essential Laboratory Techniques 2020-12, Vol.21 (1)
Hauptverfasser: Sirois, Allison R., Pilotte, Nils, Williams, Steven A., Saunders, Lori J.
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description 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
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title CRISPR/Cas9‐Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction‐Free Cloning and a Rapid Bam HI Digest Readout
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