Transcription Repression Reverses the Expected Trend of Gene-Targeting Success in Yeast

The success of gene-targeting in yeast is more efficient with anti-directional targeting modules. These modules have an insertion orientation that does not match the host gene orientation. On the other hand, targeting modules with matching orientations (i.e., codirectional) are less efficient. However, when transcription is repressed, the difference in efficiency between anti-directional and codirectional modules disappears, suggesting that transcription plays a role in shaping the gene-targeting success. An ends-out targeting strategy using the insertion-in approach was employed to target the natural GAL1 locus by the cassette pair gal1::ARG4 in an experimental setting with strongly repressed transcription in both. The putative disruptants were then selected for homologous insertion, auxanographically confirmed, and molecularly analyzed. The quantitative analysis reveals that gene-targeting in systems with repressed transcription reverses the usual trend, in which anti-directional cassettes outperform codirectional cassettes when transcription is active. At the same time, it eliminates the equal targeting ability of both cassette types in systems where the transcription is repressed only in the host gene of the module. These findings reveal a new aspect of yeast genetics, providing an opportunity to develop a novel genetic tool.