Marker-free genetic manipulations in yeast using CRISPR/CAS9 system
- 876 Downloads
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).
- Bähler J (2005) Cell-cycle control of gene expression in budding and fission yeast. Annu Rev Genet 39:69–94. https://doi.org/10.1146/annurev.genet.39.110304.095808 CrossRefPubMedPubMedCentralGoogle Scholar
- Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553. https://doi.org/10.1002/(SICI)1097-0061(199910)15:14<1541::AID-YEA476>3.0.CO;2-K CrossRefPubMedCentralGoogle Scholar
- Gruhlke MCH, Schlembach I, Leontiev R et al (2017) Yap1p, the central regulator of the S. cerevisiae oxidative stress response, is activated by allicin, a natural oxidant and defence substance of garlic. Free Radic Biol Med 108:793–802. https://doi.org/10.1016/j.freeradbiomed.2017.05.004 CrossRefPubMedPubMedCentralGoogle Scholar
- Nakamura T, Namba H, Ohmoto T et al (1995) Cloning and characterization of the Saccharomyces cerevisiae SVS1 gene which encodes a serine- and threonine-rich protein required for vanadate resistance. Gene 165:25–29. https://doi.org/10.1016/0378-1119(95)00503-X CrossRefPubMedPubMedCentralGoogle Scholar
- Zamir L, Zaretsky M, Fridman Y et al (2012) Tight coevolution of proliferating cell nuclear antigen (PCNA)-partner interaction networks in fungi leads to interspecies network incompatibility. Proc Natl Acad Sci 109:E406–E414. https://doi.org/10.1073/pnas.1108633109 CrossRefPubMedPubMedCentralGoogle Scholar