Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast.
J Cell Sci
; 136(6)2023 03 15.
Article
em En
| MEDLINE
| ID: mdl-36825467
ABSTRACT
The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Schizosaccharomyces
/
Proteínas de Schizosaccharomyces pombe
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article