Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe

Author:

Ishikawa Ken1ORCID,Soejima Saeko1,Saitoh Shigeaki1ORCID

Affiliation:

1. Institute of Life Science, Kurume University Department of Cell Biology , , Asahi-machi 67, Kurume, Fukuoka 830-0011 , Japan

Abstract

ABSTRACT 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.

Funder

Kurume University Millennium Box Foundation for the Promotion of Science

Ishibashi Foundation for the Promotion of Science

Kakihara Foundation for Science and Technology

Fukuoka Bio-valley Project

Japan Society for the Promotion of Science

Ministry of Education, Culture, Sports, Science and Technology

Publisher

The Company of Biologists

Subject

Cell Biology

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