A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9

Research output: Contribution to journalArticleScientificpeer-review

11 Citations (Scopus)
41 Downloads (Pure)

Abstract

Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site-directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.

Original languageEnglish
Number of pages13
JournalFEMS Yeast Research
Volume18
Issue number7
DOIs
Publication statusPublished - 2018

Fingerprint Dive into the research topics of 'A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9'. Together they form a unique fingerprint.

Cite this