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

Ishikawa, K ; Soejima, S ; et al.

In: Journal of cell science, Jg. 136 (2023-05-01), Heft 9

academicJournal

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.

Competing Interests: Competing interests The authors declare no competing or financial interests.

Titel:	Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.
Autor/in / Beteiligte Person:	Ishikawa, K ; Soejima, S ; Saitoh, S
Zeitschrift:	Journal of cell science, Jg. 136 (2023-05-01), Heft 9
Veröffentlichung:	Cambridge : Company of Biologists ; <i>Original Publication</i>: London., 2023
Medientyp:	academicJournal
ISSN:	1477-9137 (electronic)
DOI:	10.1242/jcs.261186
Schlagwort:	Animals Cell Proliferation Gene Knockdown Techniques Phenotype Mammals Schizosaccharomyces genetics
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, Non-U.S. Gov't Language: English [J Cell Sci] 2023 May 01; Vol. 136 (9). <i>Date of Electronic Publication: </i>2023 May 10. MeSH Terms: Schizosaccharomyces* / genetics ; Animals ; Cell Proliferation ; Gene Knockdown Techniques ; Phenotype ; Mammals Contributed Indexing: Keywords: Biotechnology; CRISPR-Cas; CRISPRi; Essential gene; Fission yeast; Knockdown; dCas9 Entry Date(s): Date Created: 20230510 Date Completed: 20230511 Latest Revision: 20230524 Update Code: 20240514

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