Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.

Transcription of heterochromatin during DNA replication regularly results in collision between DNA and RNA polymerases, and fork stalling can follow. Replication origins and silent heterochromatin, formed by RNA interference (RNAi), exhibit an alternating pattern near chromosome centromeres. Zaratiegui et al. show that RNAi displaces the RNA polymerase so that replication can be completed by the DNA polymerase. Histone-modifying enzymes travel with the replication fork, and in the absence of RNAi, stalled forks are repaired by recombination, but histone modifications are not made. Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification1 during the DNA replication phase of the cell cycle2. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes3 that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

Small interfering RNA (siRNA) guides dimethylation of histone H3 lysine-9 (H3K9me2) via the Argonaute and RNA-dependent RNA polymerase complexes, as well as base-pairing with either RNA or DNA. We show that Argonaute requires the conserved aspartate-aspartate-histidine motif for heterochromatic silencing and for ribonuclease H–like cleavage (slicing) of target messages complementary to siRNA. In the fission yeast Schizosaccharomyces pombe , heterochromatic repeats are transcribed by polymerase II. We show that H3K9me2 spreads into silent reporter genes when they are embedded within these transcripts and that spreading requires read-through transcription, as well as slicing by Argonaute. Thus, siRNA guides histone modification by basepairing interactions with RNA.