Marker-free genetic manipulations in yeast using CRISPR/CAS9 system
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The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.
KeywordsS. cerevisiae CRISPR/CAS9 Yeast Marker-free
We thank all the members of the Aharoni’s lab for advices and support. This work was supported by the Israeli Science foundation (ISF) Grant numbers 2297/15 and 1340/17, Binational Science Foundation (BSF) Grant number 2013358 and the European research training network (ITN, Horizon 2020) ES-cat (722610).
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