Soreanu Inga, Hendler Adi, Dahan Danielle, Dovrat Daniel, Aharoni Amir
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105, Be'er Sheva, Israel.
Curr Genet. 2018 Oct;64(5):1129-1139. doi: 10.1007/s00294-018-0831-y. Epub 2018 Apr 6.
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.
芽殖酵母目前是研究多种生物学过程的主要模式生物之一。酵母的基因操作涉及大量使用可选择标记，这可能会导致不良影响。因此，酵母中的无标记基因操作对于基因/启动子替换和各种其他应用非常有必要。在这里，我们结合可选择标记的优势，随后采用CRISPR/CAS9基因组编辑技术，以无标记的方式在酵母中进行常见的基因操作。我们展示了用于全基因和启动子替换以及高效操纵子阵列整合的方法。我们的方法允许利用数千种现有菌株，包括文库菌株，以无标记和无克隆的方式在酵母中产生显著的基因变化。
