Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9.
Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. Copyright © 2016 John Wiley & Sons, Ltd.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520, USA.|Nanobiology Institute, Yale University, West Haven, CT, 06516, USA.
Ronan
R
Fernandez
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520, USA. julien.berro@yale.edu.|Nanobiology Institute, Yale University, West Haven, CT, 06516, USA. julien.berro@yale.edu.
Julien
J
Berro
D000595:Amino Acid Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D005459:Fluorides / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods; D005819:Genetic Markers; D005822:Genetic Vectors / Q000145:classification / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*
D005819:Genetic Markers; D007473:Ion Channels; D005459:Fluorides
CRISPR/Cas9; Schizosaccharomyces pombe; fluoride channel; plasmid