Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest, while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineeringStreptococcus pyogenesCas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Upon recognizing complementary RNAs, the engineered sgRNAs become activated, enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimizations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAsin vivo. Our sensors open up novel opportunities for the development of new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.