A trigger-inducible split-Csy4 architecture for programmable RNA modulation

The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools. In this study, we developed a trigger-inducible split-Csy4 system to efficiently and specifically cleave RNA in mammalian cells. The dataset includes processed RNA-Seq data from HEK-293T cells expressing full-length or split Csy4, and treated with either DMSO or grazoprevir, a small molecule that triggers the reconstitution of split-Csy4. Our findings show that both the split and reconstituted forms of the split-Csy4 system had significantly less impact on the transcriptome of HEK-293T cells compared to the full-length Csy4 nuclease.