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Summary Genes embedded in H3 lysine 9 methylation (H3K9me)–dependent heterochromatin are transcriptionally silenced 1 – 3 . In fission yeast, Schizosaccharomyces pombe , H3K9me-mediated heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent 4 , 5 . Under certain conditions wild-type cells might utilize heterochromatin heritability to form epimutations, phenotypes mediated by unstable silencing rather than DNA changes 6 , 7 . Here we show that resistant heterochromatin-dependent epimutants arise in threshold levels of caffeine. Unstable resistant isolates exhibit distinct heterochromatin islands, which reduce expression of underlying genes, some of which confer resistance when mutated. Targeting synthetic heterochromatin to implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, allowing wild-type cells to adapt to non-favorable environments without altering their genotype. In some isolates, subsequent or co-occurring gene amplification events augment resistance. Caffeine impacts two anti-silencing factors: Epe1 levels are downregulated, reducing its chromatin association; and Mst2 histone acetyltransferase expression switches to a shortened isoform. Thus, heterochromatin-dependent epimutant formation provides a bet-hedging strategy that allows cells to remain genetically wild-type but adapt transiently to external insults. Unstable caffeine-resistant isolates show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to antifungal resistance in plant and human pathogenic fungi.

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6+ or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.