Abstract
Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.
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All relevant data are included in the manuscript. Requests for reagents or further information should be directed to H.T. (htsubouchi@bio.titech.ac.jp) or H.I. (hiwasaki@bio.titech.ac.jp).
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Acknowledgements
We are grateful to the Biomaterials Analysis Division, Open Facility Center, Tokyo Institute of Technology for sequence analysis. We also thank Yumiko Kurokawa and all members of the Iwasaki laboratory for stimulating discussion.
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This work was supported in part by Grants-in-Aids for Scientific Research (A) (18H03985 to H.I.), for Scientific Research (B) (18H02371 to H.T.), for Young Scientists (B) (17K15061 to B.A.), and for Early-Career Scientists (20K15713 to B.A.) from the Japan Society for the Promotion of Science (JSPS). H.T. is also supported by the Takeda Science Foundation.
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MP, HT and OB conducted experiments. Y-WH, RK and TI are responsible for sequencing analyses. MP, HT and HI are responsible for conceptualization and project design. HT, BA, YM and HI supervised the study. MP, HT and HI are responsible for data analysis. HT, BA, MP and HI wrote the manuscript.
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Palihati, M., Tsubouchi, H., Argunhan, B. et al. Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens. Curr Genet 67, 919–936 (2021). https://doi.org/10.1007/s00294-021-01201-3
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DOI: https://doi.org/10.1007/s00294-021-01201-3