Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast

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.

Automated summary

The CRISPR-Cas13d system is a promising tool for RNA manipulation, as it targets RNA with a small effector protein and does not require specific sites in the target transcript. In this study, the CRISPR-Cas13d system was successfully implemented in fission yeast for reversible RNA knockdown. The system's effectiveness was ensured by using an array of CRISPR RNAs (crRNAs) expressed from an RNA polymerase II promoter. This new toolkit is expected to have diverse applications in biology, including gene function analysis and RNA visualization.