Sensing endogenous RNA in living human cells using a CRISPR-activated protease

Most techniques used to detect specific mRNAs in eukaryotic cells require to extract nucleic acids and thereby kill the cells. A programmable sensor for monitoring endogenous transcripts in living cells, in contrast, would enable to enrich living cells based on a specific transcription or splicing event, and studying these cells by live microscopy or sequencing methods requiring intact cells. We have engineered CRISPR-READ, a live cell RNA detector based on the CRISPR-associated Lon protease CalpL and a cA ₄ -producing Type III CRISPR system. Upon RNA-programmable RNA sensing, CRISPR-READ produces an orthogonal second messenger, which leads to the cleavage of a dual FRET / localization reporter compatible with FACS sorting and live microscopy. Using this genetically encoded sensing circuit as a readout for a genome-wide CRISPR perturbation screen, we identified an extended Type-I interferon signaling cascade; RNA-Seq on sensor-sorted cells enabled unbiased identification of correlated stochasticity in gene expression across single cells.