Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here, we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here, we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.

Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.

Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3′ untranslated region (3′UTR) disruption, are available for yeast systems, CRISPR–Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR–Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.

In a chemical mechanical polishing (CMP) process, the removal rate is affected by the actual contact conditions between the wafer and the polishing pad. The polishing pad is one of the most important consumable materials: when the wafer is polished, the pad surface asperity changes. Further, the polishing pad surface asperity has a substantial influence on the actual contact conditions. Therefore, measurement and quantitative evaluation methods for the pad surface asperity have been proposed by various research institutes. We have developed a novel measurement and quantitative evaluation method for polishing pad surface asperity based on contact image analysis using an image rotation prism. We have proposed four effective evaluation parameters: the number of contact points, the contact ratio, the maximum value of the minimum spacing of the contact points, and the half-width of the peak of the spatial Fast Fourier transform (FFT) result of a contact image. This paper discusses the change in the polishing pad surface asperity measured by the proposed evaluation parameters in serial batch polishing tests. In particular, this research focused on the relationships between the proposed evaluation parameters and the removal rate, which change with an increase in the number of serial batch polishing tests. As a result, linear correlations were found between the evaluation parameters and the removal rate.

Many recent lines of evidence from the human microbiome and other fields indicate bacterial involvement in various types of cancer. Helicobacter pylori has been recognized as the major cause of stomach cancer (gastric cancer), but the mechanism by which it destabilizes the human genome to cause cancer remains unclear. Our recent studies have identified a unique family of toxic restriction enzymes that excise a base (A: adenine) from their recognition sequence (5′-GTAC). At the resulting abasic sites (5′-GT_C), its inherent endonuclease activity or that of a separate endonuclease may yield atypical strand breaks that resist repair by ligation. Here, we present evidence demonstrating involvement of its H. pylori member, HpPabI, in stomach carcinogenesis: (i) Association of intact HpPabI gene with gastric cancer in the global H. pylori Genome Project and the open genomes; (ii) Frequent mutations at A in 5′-GTAC in the gastric cancer genomes as well as in H. pylori genomes; (iii) Its induction of chromosomal double-strand breaks in infected human cells and of mutagenesis in bacterial test systems. In addition, its unique regions that interact with DNA exhibit signs of diversifying selection. Our further analysis revealed similar oncogenic bacterium–restriction–enzyme pairs for other types of cancer. These results set another stage for cancer research and medicine around oncogenic restriction enzymes.

Summary: The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism to study essential biological processes. Its genome contains ∼1200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe , using a dCas9-mediated CRISPRi. These libraries cover ∼98% of all essential genes in fission yeast. We estimate that in ∼60% of the strains in the library, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed a flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate understanding of essential biological processes in eukaryotes.