期刊
JOURNAL OF CELL SCIENCE
卷 136, 期 9, 页码 -出版社
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.261186
关键词
Knockdown; Essential gene; CRISPR-Cas; CRISPRi; dCas9; Fission yeast; Biotechnology
类别
This study utilized CRISPRi technology to study conserved essential obscure (ceo) genes from yeasts to mammals. By using dCas9-mediated CRISPRi and enhancing repression efficiency, the transcription of target genes was successfully reduced, allowing the characterization of resulting phenotypes. This study demonstrated that dCas9-based CRISPRi methods enhance accessibility of genetic analyses targeting essential genes 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据