Journal
G3-GENES GENOMES GENETICS
Volume 11, Issue 4, Pages -Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/g3journal/jkab051
Keywords
knockdown; transcription; CRISPR-Cas; CRISPRi; dCas9; fission yeast; biotechnology
Categories
Funding
- Japan Society for the Promotion of Science [17K07394, 20K06648]
- MEXT-Supported Program for the Strategic Research Foundation at Private University from the Ministry of Education, Culture, Sports, Science and Technology, Japan
- Grants-in-Aid for Scientific Research [20K06648, 17K07394] Funding Source: KAKEN
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Controllable and reversible transcriptional repression using CRISPRi technology with dCas9 and sgRNA has been successfully implemented in fission yeast, achieving up to 87% repression of endogenous gene transcription and altering cellular phenotypes.
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.
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