CRISPR-Cas9 editing efficiency in fission yeast is not limited by homology search and is improved by combining gap-repair with fluoride selection.

Protocols for CRISPR-Cas9 editing have been implemented in most model organisms, including fission yeast, for which some improvements have also been later described. Here, we report an improvement to the CRISPR-Cas9 protocol in fission yeast, as we combine a cloning free gap-repair method with our previously described fluoride selection marker, which speeds up genome editing. We also report a wide variability of editing efficiencies at different loci along the genome, and we demonstrate that this variability cannot be explained by the location of the edited sequences in the genome. Lastly, our attempt at improving editing efficiency by targeting the donor DNA to the cut site using a HaloTag strategy to link the donor DNA to two proteins of the homologous recombination repair machinery ( Rad51 or Rad52 ) fell short, which shows that editing efficiency in fission yeast is likely not limited by homology search.

Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

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.

A transposon-induced epigenetic change leads to sex determination in melon.

Sex determination in plants leads to the development of unisexual flowers from an originally bisexual floral meristem. This mechanism results in the enhancement of outcrossing and promotes genetic variability, the consequences of which are advantageous to the evolution of a species. In melon, sexual forms are controlled by identity of the alleles at the andromonoecious (a) and gynoecious (g) loci. We previously showed that the a gene encodes an ethylene biosynthesis enzyme, CmACS-7, that represses stamen development in female flowers. Here we show that the transition from male to female flowers in gynoecious lines results from epigenetic changes in the promoter of a transcription factor, CmWIP1. This natural and heritable epigenetic change resulted from the insertion of a transposon, which is required for initiation and maintenance of the spreading of DNA methylation to the CmWIP1 promoter. Expression of CmWIP1 leads to carpel abortion, resulting in the development of unisexual male flowers. Moreover, we show that CmWIP1 indirectly represses the expression of the andromonoecious gene, CmACS-7, to allow stamen development. Together our data indicate a model in which CmACS-7 and CmWIP1 interact to control the development of male, female and hermaphrodite flowers in melon.

A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons.

Andromonoecy is a widespread sexual system in angiosperms characterized by plants carrying both male and bisexual flowers. In melon, this sexual form is controlled by the identity of the alleles at the andromonoecious (a) locus. Cloning of the a gene reveals that andromonoecy results from a mutation in the active site of 1-aminocyclopropane-1-carboxylic acid synthase. Expression of the active enzyme inhibits the development of the male organs and is not required for carpel development. A causal single-nucleotide polymorphism associated with andromonoecy was identified, which suggests that the a allele has been under recent positive selection and may be linked to the evolution of this sexual system.