How to Turn Negative into Positive: Application of CRISPR Endoribonuclease Csy4 As a Negative Operator to Detect Negative Signals in Planta

The CRISPR Class 1 endoribonuclease Csy4 from Pseudomonas aeruginosa binds and cleaves the repetitive sequence of the pre-crRNA. The Csy4 target sequence (C4T) can be integrated into reporter genes (like GUS or Venus) making them Csy4-sensitive on the RNA-level. In planta studies revealed that Csy4 can completely block reporter gene expression. This served as basis for the investigation of RNA-degrading and DNA-editing tools targeting Csy4. This system allows to convert a gene-repressing component into a positive signal. Thus, the optimization of these repressing components can be carried out by monitoring the extent of de-repression of the reporter. Here, properties and applications of the Csy4-based reporter system will be presented.