Abstract
Gene-targeting is one of the most important molecular tools for genomic manipulations for research and industrial purposes. However, many factors influence targeting fidelity undermining the efforts for accurate, fast, and reliable construction of genetically modified yeast strains. Therefore, it is of great academic interest that we uncover as many as possible parameters affecting the recombination mechanisms that enable targeting. Since usually, researchers choose the orientation of the insertion (marker) within the module at random, it seemed interesting to see whether the same module will achieve essentially the same targeting efficiency when the same marker was oriented alternatively concerning the same target gene. Thus, two loci (URA3 and LEU2) and one allele (ura3-52) in a haploid yeast genetic background were targeted by artificial modules bearing homologous insertions in alternative orientations being flanked by long asymmetrical targeting homology to either replace or disrupt a genomic target. Results showed that insertion orientation within the targeting module strongly influences targeting in yeast, regardless of the targeting approach.
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Abbreviations
- HR:
-
Homologous recombination
- EOGT:
-
Ends-out gene-targeting
- NHEJ:
-
Non-homologous end-joining
- BIR:
-
Break-induced DNA replication
- FH:
-
Flanking homology
- THet:
-
Terminal heterology
- GTC:
-
Gene-targeting cassette
- TS:
-
Targeting success
- TC:
-
Targeting coefficient
- rQTE:
-
Relative quantitative targeting efficiency
- TGE:
-
Targeted genetic event
- nTGE:
-
Non-targeted genetic event
- YTE:
-
Yeast transformation efficiency
- SC:
-
Synthetic complete media
- ds:
-
Double-stranded
- nt:
-
Nucleotide(s)
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Acknowledgements
This article is dedicated to the living memory of Professor Zoran Zgaga (1956-2011) under whose mentorship these experiments were conducted. I thank Dr. Alan Nicolas and Associate Professor Tomislav Domazet-Lošo for the courtesy of helping me with the genetic background of the ORD-2-7B strain and for providing the PCR primers, respectively. Special thanks go to the tragically deceased Mrs. Nataša Tomašević (1971-2020) and Mr. Vlado Bartovsky for skillful technical assistance. I also thank Dr. Mary Sopta for proofreading the manuscript.
Funding
This research was funded by grant 0058014 from the Croatian Ministry of Science, Education, and Sports to Zoran Zgaga.
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This research was conceptualized by ZZ (see Acknowledgments; replacement-of approach) and PTM (deletion-from and insertion-in approaches), the methodology was chosen by ZZ and PTM, formal analysis, investigation, data curation was executed by PTM, writing, and visualization were conceptualized by PTM, and funding acquisition was done by ZZ.
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Mitrikeski, P.T. Insertion orientation within the cassette affects gene-targeting success during ends-out recombination in the yeast Saccharomyces cerevisiae. Curr Genet 68, 551–564 (2022). https://doi.org/10.1007/s00294-022-01246-y
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DOI: https://doi.org/10.1007/s00294-022-01246-y