Sensing and perturbing mammalian cell states with reprogrammable ADAR sensors (RADARS)

Abstract

Reprogrammable Adenosine Deaminase Acting on RNA (ADAR) Sensors (RADARS) control RNA translation in mammalian cells, allowing for noninvasive sensing or perturbation of specific cell types based on transcriptional signatures. Upon base-pairing between a target RNA and a sensor RNA, RADARS leverages ADAR to edit a premature stop codon upstream of a gene of interest, thereby releasing translation of the desired cargo. These design principles enable sequence programmability, allowing RADARS to adapt more easily to new contexts than existing tools for targeting cell types. We describe a detailed protocol for performing experiments with RADARS, including designing, cloning and validating RADARS constructs targeting a transcript of interest. RADARS guide sequences can be designed with an intuitive web interface and cloned into existing constructs for downstream applications including imaging, sorting and sequencing. We outline recommendations for cargo choice, sensor design and ADAR system selection, enabling users to choose the best workflow depending on the desired application. Beginning with sensor design, the selection of top-performing RADARS guides can be completed in ~2 weeks, followed by a desired use case. Convenient engineering and application of RADARS for various applications enable the design and execution of various cell-targeting experiments. This protocol uses Reprogrammable Adenosine Deaminase Acting on RNA (ADAR) Sensors (RADARS) to robustly sense RNA transcripts inside eukaryotic cells, enabling detection of changes in gene expression or targeting and perturbation of specific mammalian cell types and states.