Marker-free genetic manipulations in yeast using CRISPR/CAS9 system

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.